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Appendices 1-18 to: Report on the Health Effects of Selected Pesticide Coformulants

99     Animal toxicity

99.1     Single dose toxicity

99.1.1     Inhalation

No rats died when exposed for 7 hours to the maximum attainable vapour concentration (estimated by DECOS to be 122 mg/m³) of DEGBE (Gingell et al. 1994 – quoted from DECOS 1996 and from ECETOC 1995a).

Also rabbits, cats, guinea pigs, rats, and mice survived an exposure period of 2 hours in a saturated atmosphere (DECOS 1996).

An LC₅₀-value of about 73000 mg/m³ has been reported for rats following exposure for 4 hours to the acetate (DEGBEA) of DEGBE. Signs of toxicity were red foci and congestion in the lungs. (DuPont 1984 – quoted from ECETOC 1995b).

99.1.2     Oral intake

The oral LD₅₀-values reported for DEGBE are between 5080 and 9600 mg/kg b.w. in rats (6 values reported), between 2400 and 6560 mg/kg b.w. in mice (3 values reported), 2200 mg/kg b.w. in rabbits, and 2000 mg/kg b.w. in guinea pigs (Quoted in DECOS 1996, A&H 1995, ECETOC 1995a, and in IUCLID 2000).

99.1.3     Dermal contact

The dermal LD₅₀-value reported for DEGBE is greater than 2000 mg/kg b.w. for rats, and between 2700 and 4120 mg/kg b.w. for rabbits (2 values reported) (Quoted in DECOS 1996, A&H 1995, ECETOC 1995a, and in IUCLID 2000).

99.1.4     Skin irritation

In a skin irritation study in rabbits performed according to directive 84/449/EEC, B.4 “Acute toxicity (skin irritation)”, very slight to moderate erythema and oedema was observed 60 minutes following the application of DEGBE. After 3 days, only very slight irritation was observed and after 9 days, all symptoms had disappeared. (Southwood 1987 - quoted from IUCLID 2000).

DEGBE has been reported to be only very slightly irritating to the skin of rabbits (Rowe et al. 1982 – quoted from DECOS 1996; Gingell et al. 1994 – quoted from ECETOC 1995a) and slightly irritating to the skin of rabbits and guinea pigs (Krasavage & Terhaar 1981 – quoted from Gingell et al. 1996).

99.1.5     Eye irritation

99.1.5.1     Eye irritation

In a primary eye irritation test, 0.1 ml of undiluted or diluted (1, 5, 10, 20, 25, or 50% in water) DEGBE was instilled into the eyes of rabbits (6 animals per group). The eyes were examined after 10 minutes, 1 hour, 24 hours, and daily up to 14 days. Instillation of 1% DEGBE caused a 3% increase in intraocular pressure after 10 minutes, and 20% DEGBE caused a 63% increase; the values returned to normal within an hour. Instillation of 10% DEGBE caused a mild, transient conjunctivitis, and 25% caused increased tear flow, congestion, inflamed eyelids (blepharitis), conjunctival oedema (chemosis), and corneal inflammation (keratitis); these effects disappeared within 2 days. Instillation of 50 and 100% DEGBE caused iritis as well, which lasted 3 and 10 days, respectively. (Ballantyne 1984 – quoted from A&H 1995, DECOS 1996, ECETOC 1995a, and IUCLID 2000).

Following instillation of 0.005 ml of undiluted DEGBE into the eyes of rabbits and observation for 18 to 24 hours, moderate irritation and corneal damage were recorded, graded 5 on a scale of increasing damage 1 to 10 (Smyth & Carpenter 1946 – quoted from IUCLID 2000 and from A&H 1995).

DEGBE has been reported to be capable of causing moderate irritating and moderate transient corneal injury to the eyes of rabbits (Rowe et al. 1982 – quoted from DECOS 1996).

99.1.6     Sensitisation

In a guinea pig maximization test performed according to directive 84/449/EEC, B.6 “Acute toxicity (skin sensitization)”, 10 guinea pigs were induced with 2.5% of the test substance (intradermally) or with the undiluted test substance (covered patch). For the challenge, undiluted test substance was used. The control group consisted of 4 animals. No skin sensitisation was observed. (Basketter 1985 – quoted from IUCLID 2000).

DEGBE was not sensitising in the guinea pig maximisation test (25% injection induction, 100% application induction and application challenge) (Unilever 1984 – quoted from ECETOC 1995).

99.2     Repeated dose toxicity

99.2.1     Inhalation

When rats (20 animals per group) were exposed to DEGBE at concentrations of 0 or 143 mg/m³, 6 hours a day for 4 days, no changes were induced in clinical chemistry, haematology parameters, or in histology; no further details were given (DFG 1992 – quoted from DECOS 1996).

Wistar rats (5 animals of each sex per group) were exposed (unclear whether whole-body or nose-only) to DEGBE at concentrations of 0, 100, 350, or 1000 mg/m³, 6 hours a day, 5 days per week for 2 weeks. According to IUCLID, 100 mg/m³ is the highest attainable vapour concentration. The body weights were decreased at the highest concentration, but not significantly. A dose-related decrease in spleen weight was noted in all male exposure groups and increased (but not dose-related) lung weights at the two highest concentrations. Histopathology revealed perivascular and peribronchial accumulations of granulocytes and activation of alveolar epithelium at all exposure levels. The NOAEL was considered to be below 100 mg/m³. (BASF 1987 – quoted from IUCLID 2000).

In a study performed according to OECD Guideline 412, Wistar rats (10 females per group) were exposed (head-nose) to DEGBE at concentrations of 0 or 350 mg/m³, 6 hours a day, 5 days per week for 2 weeks. Satellite groups were observed for 4 weeks post-exposure. No effects on mortality, organ weights, clinical chemistry, and gross pathology were observed. The body weight gain was significantly decreased in both main and satellite groups. Very slight nasal discharge was observed in some animals during exposure. Histopathology revealed slight to moderate multifocal perivascular and peribronchial accumulation of granulocytes and minimal focal bronchiolisation (no further details are given) in the lungs. After 4 weeks, recovery was observed regarding all effects except minimal to slight granulocytes accumulation in the lungs (regression in severity was evident). The NOAEL was considered to be below 350 mg/m³. (BASF 1991 – quoted from IUCLID 2000).

Fischer 344 rats (15 animals of each sex per group) were exposed (whole-body) to DEGBE (vapour) at concentrations of 0, 2, 6, or 18 ppm (0, 13, 40, or 122 mg/m³, 6 hours a day, 5 days per week for 5 weeks. No treatment-related findings were observed regarding mortality, body weights, clinical signs, haematology, and urinalysis. A significantly decreased relative liver weight was observed in males at the two highest exposure levels (dose-related) whereas a dose-related increase was observed in females at these exposure levels (significant only at the highest exposure level). In female rats, slight paleness of the liver was noted in 3/10 animals at the highest exposure level and slight hepatocyte vacuolisation at all dose levels (3/10, 4/10, 9/10, 10/10 of the control, low-, mid-, and high-dose groups, respectively). According to the authors, the slight hepatocytes vacuolisation in females was consistent with fat accumulation and was considered to be of questionable toxicological significance. The NOAEL was considered to be 13 mg/m³ (DECOS) or 40 mg/m³ (IUCLID, the decreased liver weight in mid-dose males were not accompanied by histological changes and thus considered not to be treatment-related). (Gushow et al. 1981 – quoted from IUCLID 2000, ECETOC 1995a, and from DECOS 1996).

In a study performed according to OECD Guideline 413, Wistar rats (10 animals of each sex per group in either the main groups and the satellite groups) were exposed (whole-body) to DEGBE at concentrations of 0, 2, 6, or 14 ppm (0, 13, 40, or 95 mg/m³), 6 hours a day, 5 days per week for 13 weeks. Satellite groups were observed for 4 weeks post-exposure. No mortality and no treatment-related findings were observed for body weight (change), clinical signs, ophthalmology, haematology, urinalysis, organ weights, and pathology; according to IUCLID, no data on spleen weights were reported. The only observed effect was a dose-related decreased level of serum aspartate aminotransferase in males of all exposure groups; this change was, according to IUCLID, not considered as being treatment-related because no effects on liver were observed and the change was within the range of biological variation. Therefore, the NOAEL was considered to be 95 mg/m³; DECOS has endorsed this conclusion. (BASF 1992 – quoted from IUCLID 2000, Gingell et al. 1996, and from DECOS 1996).

99.2.2     Oral intake

DEGBE was fed to rats (5 animals of each sex per group) at dose levels ranging from 51 to 1830 mg/kg b.w./day for 30 days (according to Gingell et al. (1996), the study is a drinking water study). The lowest dose level that caused loss of appetite was 94 mg/kg b.w./day, and at 650 mg/kg b.w./day histopathological changes were observed in either the liver, kidney, spleen, or testes (no details are given of which organs were affected). (Smyth & Carpenter 1948 – quoted from A&H 1995, ECETOC 1995a, DECOS 1996, and from Gingell et al. 1996).

The NOAEL was considered, by ECETOC, to be 51 mg/kg b.w./day.

According to Gingell et al. (1996), DEGBE, in the 1940s, was made by a process using boron trifluoride as a catalyst and may have contained impurities, which could have caused the observed effects.

Sprague-Dawley rats (10 males per group) received 0, 891, 1782, or 3564 mg/kg b.w./day of DEGBE by oral gavage, 5 days a week for 6 weeks. The control group received the vehicle (not specified). Feed consumption and body weight were decreased at the highest dose level and congestion of the spleen was observed. A dose related decrease in haemoglobin, total red cells, mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), and mean corpuscular haemoglobin concentration (MCHC); increase in absolute and relative spleen weights; decrease in liver weight; and proteinaceous casts were observed at the mid- and high-dose levels. Hyperkeratosis in the stomach was observed in all dosed rats. The NOAEL was lower than 891 mg/kg b.w./day. (Eastman Kodak Company 1984 – quoted from IUCLID 2000). This study is also cited in Gingell et al. (1996); however, not every of the abovementioned effects are included in this citation.

Fischer 344 rats (16 animals of each sex per group for six weeks and 10 for the rest of the study) received oral doses of DEGBE by gavage of 0, 65, 327, or 1630 mg/kg b.w./day (males) or 0, 51, 254, or 1270 mg/kg b.w./day (females) 5 days a week for 13 weeks. At the highest dose level, 83/92% of male/female rats died within the study with 60/30% at the mid-dose level and 0/10% at the low dose level; pulmonary congestion and oedema was observed in the rats that died during the study and, according to ECETOC and Gingell et al., the very high mortality was due to dosing accidents. Due to the high mortality in the high-dose group, statistics were not performed at this dose levels. Food consumption (males and females) and body weight gain (males) were decreased at the highest dose level. Absolute and relative liver weights were increased in the mid-dose males and, according to IUCLID, in the remaining males and females of the high-dose group; the relative kidney weight was, according to IUCLID, increased in the remaining high-dose males; and the relative spleen weight was, according to DECOS, decreased in mid-dose male animals. Haematological changes were found in both males (according to DECOS only: increased erythrocyte count and haemoglobin content at the mid-dose level) and females (dose-related decreased leukocyte and lymphocyte count, and mean corpuscular haemoglobin concentration at the two lowest dose levels). Gross and microscopic lesions were, according to IUCLID, only observed in the thoracic cavity and respiratory tract. (Hobson et al. 1987 – quoted from IUCLID 2000 and from DECOS 1996). This study is also cited in Gingell et al. (1996) and in ECETOC (1995a); however, the results of the study are very briefly reported in these two sources.

99.2.3     Dermal contact

Sprague-Dawley rats (10 animals of each sex per group) received 2 ml/kg b.w./day of 0, 10, 30 or 100% of DEGBE in water (equal to 0, 200, 600 or 2000 mg/kg b.w./day) by application to the skin (3 cm by 3 cm area on the clipped skin of the back) under occlusion for 6 hours a day, 5 days per week for 13 weeks. No mortality and no treatment-related findings were observed for feed consumption, body weights, clinical chemistry, haematology, organ weights, oestrous cycling in females, and pathology. The only suggestion of a systemic effects was a slightly increased incidence of occult blood in the urine of females in the two highest dose groups; microscopic examination of the urine revealed no urinary casts and no significant numbers of erythrocytes. DEGBE produced skin irritation, which was dose-concentration dependent in incidence, severity, and time of onset and was more severe in females than in males; irritation was minimal at the lower two concentrations whereas application of undiluted DEGBE produced irritation in all animals, some were affected after as few as two doses. Microscopic examination of skin sections from the application site revealed no DEGBE-related histological changes, slight thickening of the epidermis being seen for both control and treated animals. A NOAEL of 2000 mg/kg b.w./day can be considered for systemic effects; for local effects the NOAEL is below 200 mg/kg b.w./day. (Auletta et al. 1993).

In a study performed in accordance with the US-EPA test guidelines for neurotoxicity, Sprague-Dawley rats (12 animals of each sex per group) received 2 ml/kg b.w./day of 0, 10, 30 or 100% of DEGBE in water (equal to 0, 200, 600 or 2000 mg/kg b.w./day) by application to the skin (the test volume was applied to the dorsum and spread over the previously shaven area, approximately 10% of the body surface) under occlusion for 6 hours a day, 5 days per week for 13 weeks. Male and female rats were examined using a functional observational battery (FOB) pre-study, at 1, 6, and 24 hours after the initiation of the first exposure, and prior to treatment on days 7, 14, 35, 63, and 91. Motoractivity was determined pre-study and on non-treated days 34, 62, and 90. At the completion of the treatment, animals from the highest dose level and 6 animals from the control group were perfused for neuropathology. There was no mortality and feed consumption and body weights were unaffected by treatment. Five females at the highest dose level had scab formation at the treatment site during the study; no other treatment-related clinical findings were noted. The FOB and motor activity tests revealed no findings indicative of a neurotoxic effect, and there were no gross or neuropathological treatment-related changes. A NOAEL of 2000 mg/kg b.w./day can be considered for systemic effects, including neurotoxicity; the NOAEL for local effects was 600 mg/kg b.w./day. (Beyrouty et al. 1993).

New Zealand white rabbits (3 animals of each sex per group) had 2 ml/kg b.w./day of 0 or 1.5% of DEGBE in water (equal to 0 or 30 mg/kg b.w./day) applied on abraded skin without occlusion for 7 hours a day, 5 days per week for 4 weeks. Oral exposure was prevented by collars. There were no treatment-related effects regarding clinical signs, urinanalysis, organ weights, and gross pathology. The water consumption was decreased in males, and haematological (decreased eosinophil and monocyte count in males; decreased haemoglobin, erythrocyte, leukocyte and neutrophil count in females) and clinical chemical (increased alkaline phosphatase in females, but decreased alkaline phosphatase in males; decreased globulin and sodium level in females) changes were observed; these effects were, according to IUCLID, not considered treatment-related because of a wide variation within one group and/or between the groups before and after exposure. No treatment-related effects on the skin were noted. A NOAEL of 30 mg/kg b.w./day can be considered for systemic and for local effects. (Elliot et al. 1982 – quoted from IUCLID 2000).

99.3     Toxicity to reproduction

99.3.1     Inhalation

No data have been found.

99.3.2     Oral intake

In a one-generation study, male Charles River CD rats Groups (25 animals per group) were dosed orally by gavage with 0, 250, 500 or 1000 mg/kg b.w./day of DEGBE for 60 days prior to mating and until sacrifice. Female rats (25 animals per group) were treated similarly for 14 days prior to mating and until sacrifice (one-half of each dose group at gestation day 13 and the reaming animals at lactation day 21). Untreated males were mated with treated females and vice versa. Ten animals died during the study; deaths were attributed to intubation accidents or to the aspiration of the test substance. The numbers of fertile males in the treated groups were not significantly different from controls, nor were there any significant increases in resorptions or decreases in live embryos and live births in untreated females mated with treated males. No significant differences were seen in the body weights of the treated females during the study, and there were no significant differences in the number of fecund females or in resorptions, live embryos, and live pups at birth. The only significant effect observed was a decrease in the weight of the pups at day 14 of lactation in offspring from the high-dose female group. A NOAEL of 1000 mg/kg b.w./day can be considered for reproductive and developmental effects and of 500 mg/kg b.w./day for effects in the offspring (reduced pup weight ). (Nolen et al. 1985).

Wistar rats (20 female per group) received 0, 0.04, 0.2, or 1% of  DEGBE in their diet (equal to 0, 25, 115, or 633 mg/kg b.w./day) from day 0 to 20 of gestation. On day 20 of gestation, 14-15 rats in each group were sacrificed; the remainder of the rats were allowed to deliver spontaneously and to rear their offspring until weaning where they were sacrificed, the offspring were sacrificed 10 weeks after birth. No deaths or clinical signs of toxicity were observed and the feed consumption in treated animals was similar to that of the control group. The maternal body weight gain was significantly reduced in all exposure groups during the gestation period. No significant differences were found in the pre- and postimplantation losses, the numbers of corpora lutea per litter, implantations per litter, the number of live foetuses per litter, the sex ratio of live foetuses, and the foetal body weight. External, skeletal, and internal examinations of the foetuses revealed no evidence of teratogenesis. In the postnatal period, a high survival rate and good growth of the offspring were noted. A NOAEL of 633 mg/kg b.w./day can be considered for developmental effects, including teratogenicity. (Ema et al. 1988).

When CD-1 mice (46-48 animals per group) were exposed orally by gavage to 0, 500 or 2050 mg/kg b.w./day of DEGBE from day 7 to 14 of gestation, no indications for developmental effects were observed; however, routine examinations of pups for malformations or skeletal anomalies were, according to IUCLID, not performed. At the high dose, 25% of the dams died. At the low dose, there were no maternal deaths and body weight was similar to controls. (Hardin et al. 1987 - quoted from IUCLID 2000 and from DECOS 1996).

99.3.3     Dermal contact

In a one-generation study, Sprague-Dawley rats (25 animals of each sex per group) received 2 ml/kg b.w./day of 0 or 100% of DEGBE in water (equal to 0 or 2000 mg/kg b.w./day) by application to the skin (3 cm by 3 cm area on the clipped skin of the back) under occlusion for 6 hours a day, 5 days per week for 13 weeks. The animals were then mated within dose groups. The males continued to be treated for 6 hours a day, 5 days per week, until the completion of the mating period. The females were treated for 6 hours a day, 7 days per week during the mating period and on gestational days 0 to 20, and were sacrificed after day 21 of lactation. No mortality and no treatment-related findings were observed for feed consumption and body weights. The male and female mating indices, pregnancy rates, male fertility indices, along with parturition data, pup body weights, and pup survival and viability were not adversely affected by treatment with 100% DEGBE. Examinations of the pups delivered by treated females, pups found dead during lactation, and of the pups at weaning revealed no adverse effects due to the treatment with DEGBE. A NOAEL of 2000 mg/kg b.w./day can be considered for reproductive and developmental toxicity. (Auletta et al. 1993).

In a teratogenicity study performed according to OECD-guideline 414, New Zealand white rabbits (17-19 pregnant females per group) had 0, 100, 300 or 1000 mg/kg b.w. of DEGBE applied to the backs (10 x 20 cm on the dorsal surface, without occlusion) and left for 4 hours a day, on days 7 to 18 of gestation. Oral ingestion was prevented by plastic collars. None of the dams died during the study. There was one abortion and one early delivery, which, according to the authors, not appeared to be related to treatment with DEGBE. All of the dosed dams gained less weight than the controls during gestation, although the reduction was only significant for the mid-dose group. At the two higher dose levels, skin irritation was observed after about 1 week of treatment and persisted until the dams were sacrificed. No significant differences were seen in any of the reproductive parameters (number of corpora lutea, implants, resorptions, or viable embryos), or in the mean foetal body weight. Furthermore, there were no significant differences in the incidence of skeletal anomalies or of gross or visceral malformations. A NOAEL of 1000 mg/kg b.w./day can be considered for developmental toxicity, including teratogenicity; the NOAEL for maternal effects was 100 mg/kg b.w./day (reduced weight gain, skin irritation). (Nolen et al. 1985).

99.4     Mutagenic and genotoxic effects

99.4.1     In vitro studies

DEGBE showed negative results when tested in the Ames test in Salmonella typhimurium strains TA98, TA100, TA1535, TA1537, and TA1538 with and without metabolic activation at concentrations up to toxicity (20 µl/plate) (Thompson et al. 1984 – quoted from IUCLID 2000, A&H 1995, ECETOC 1995a, Gingell et al. 1996, and from Gollapudi et al. 1993).

DEGBE was negative in a forward gene mutation assay at the HGPRT locus of Chinese Hamster Ovary (CHO) cells (OECD guideline 476) when tested up to a maximum concentration of 5000 µg/ml with and without metabolic activation system (Gollapudi et al. 1993).

When DEGBE was tested for chromosome aberrations in CHO cells, the number of aberrations per cell, including chromatid gaps, were similar to that of negative and solvent controls. A cytotoxicity test was performed with concentrations from 1.06 to 10.56 µl/ml with and without metabolic activation; the toxicity curve was much steeper in the presence of S9 mix and doses of 4.46, 5.94, and 7.92 µg/ml were selected for the cytogenicity assay. (Thompson et al. 1984 – quoted from IUCLID 2000, A&H 1995, ECETOC 1995a, Gingell et al. 1996, and from Gollapudi et al. 1993).

DEGBE also showed a negative result when tested for unscheduled DNA synthesis (UDS) in rat hepatocytes at concentrations from 0.26 to 4.44 µg/ml without metabolic activation. Viability was determined over dose ranges from 0.26 to 10.0 µg/ml; survival figures at 4.44, 6.67, and 10.0 µg/l were 59, 32, and 2%, respectively, relative to solvent control. (Thompson et al. 1984 – quoted from IUCLID 2000, A&H 1995, ECETOC 1995a, Gingell et al. 1996, and from Gollapudi et al. 1993).

A weak, dose-dependent increase in mutations was seen in the mouse lymphoma test (L5178Y) without, but not with, metabolic activation; there was no increase at non-toxic dose-levels. The test was performed with concentrations from 0.42 to 5.6 µg/l without metabolic activation and from 0.56 to 7.5 µg/ml with metabolic activation (S9). The highest concentration used caused 88% toxicity in the absence of S9 and 42% toxicity in the presence of S9. (Thompson et al. 1984 – quoted from A&H 1995, IUCLID 2000, ECETOC 1995a, Gingell et al. 1996, and from Gollapudi et al. 1993).

99.4.2     In vivo studies

DEGBE has been evaluated in the in vivo mouse bone marrow micronucleus test for cytogenetic damage. DEGBE was administered by oral gavage at dose levels of 0, 330, 1100, or 3300 mg/kg b.w. to CD-1 mice (5 animals of each sex per group). According to the authors, the highest dose level was approximately 80% of the estimated LD₅₀-value. Animals were sacrificed 24, 48, or 72 hours after treatment. DEGBE was ineffective in increasing the incidence of micronucleated polychromatic erythrocytes. (Gollapudi et al. 1993). According to IUCLID (2000), the study was performed according to OECD-guideline 474.

DEGBE was negative when tested for lethal mutations in the Drosophila sex-linked recessive lethal assay following administration of DEGBE in the feed (11000 ppm) for 3 days or by a single injection (14000 ppm) (Thompson et al. 1984 – quoted from IUCLID 2000, A&H 1995, ECETOC 1995a, Gingell et al. 1996, and from Gollapudi et al. 1993).

99.5     Carcinogenic effects

No data have been found.

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