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

107     Animal toxicity

107.1     Single dose toxicity

107.1.1     Inhalation

Rats exposed to an atmosphere of aerosols and vapour of 500 ppm (3080 mg/m³) for 7 hours showed mild narcosis (Rowe et al. 1954).

107.1.2     Oral intake

An LD₅₀ of 5221 mg/kg (5.54 ml/kg) in rats was established in an experiment including 9 dosage levels and a total of 169 animals (Rowe et al. 1954). Another study reported an LD₅₀-value of 5.13 g/kg in rats (Smyth et al. 1962).

In dogs, the LD₅₀-value was calculated to 7500 mg/kg. Respiratory paralysis was seen. Mortality occurred within 48 h. (Shideman & Procita 1951 - quoted from ECETOC 1995).

107.1.3     Dermal contact

Rabbits were exposed to DPGME under occluded patch for 24 hours to 10, 15 or 20 ml/kg (9.4, 14.1 or 18.8 g/kg) of the substance. No deaths occurred, but slight body weight loss at all dose levels and transient narcosis was observed in the high dose group. (Rowe et al. 1954).

An LD₅₀-value of 10 ml/kg b.w. (9.4 g/kg) was calculated from the result of exposure to DPGME of 4 male albino New Zealand white rabbits under occluded patch for 24 hours (Smyth et al. 1962).

4.1.5 Skin irritation

Data from different studies reported in IUCLID indicate that the substance is not irritating to the skin of rabbits following short time open application of 95 or 500 mg/kg. No further details are provided. (IUCLID 2000).

4.1.6 Eye irritation

One drop (approx 47 mg) undiluted DPGME applied to the eyes of rabbits 5 times with an observation period of 2 weeks produced a mild transitory irritation of the conjunctiva with no cumulative effect nor corneal injury (Rowe et al. 1954).

The substance was reported to score a grade 2 for corneal injury on a

10-grade-scale following an application of 0.5 ml (470 mg) undiluted DPGME to the eyes of rabbits (Smyth et al. 1962).

Application of 0.1 ml (94 mg) DPGME to the eye of 6 female rabbits caused moderately severe conjunctivitis and conjunctoblepharitis. The effect peaked after about 6 hr and disappeared within a week. Minor keratitis occurred between the first and third day. A 40% solution (37.6 mg) instilled in the eye of 6 additional rabbits produced a mild conjunctival irritation, and a 20% solution (18.8 mg) was without effects in the third group of 6 rabbits. Intraocular tension measured in 60 rabbits and corneal thickness measured in 54 animals increased temporarily in a dose-related way, returning to normal within 3 days. (Ballantyne 1983-84).

107.1.4     Skin sensitisation

No data were found.

107.2     Repeated dose toxicity

107.2.1     Inhalation

In an unpublished range-finding study, 5 male and 5 female Fischer 344 rats and an unreported number of B6C3F1 mice of both sexes were exposed to 0, 50, 140 or 330 ppm (0, 308, 862 or 2033 mg/m³) DPGME 6 hours/day, 5 days/week for 2 weeks. No treatment-related effects were reported with respect to post-exposure clinical observations, body weights and urinalysis in either species. Absolute liver weights in male rats in the high dose-group were significantly higher than in the concurrent controls. Relative liver weights were significantly increased in male rats at all exposure levels compared to control means (7, 7 and 16% in the low-, mid- and high-dose groups, respectively). Relative liver weights were also significantly increased in female mice. However, the authors evaluated the findings not to be significant adverse effects since no gross or histopathological changes were found in the liver. (Landry et al. 1981 - quoted from Landry et al. 1984).

Groups of 10 male and 10 female Fischer 344 rats were exposed 6 hours/day, 5 days/week for 13 weeks to concentrations of 0, 15, 50 or 200 ppm (92.4, 308 or 1232 mg/m³) DPGME. There were no clinical effects related to treatment with DPGME. Rat livers exposed to DPGME at gross observation appeared to be enlarged. However, the group liver weight means did not support this observation. Histopathological examination showed only changes considered by the authors to be minimal and spontaneous and not treatment related. Details on histopathology was not reported in the article. Clinical chemistry, haematology and urinalysis parameters were not affected by treatment with DPGME. (Landry et al. 1984).

The above-mentioned study included investigation in groups of 7 male and 7 female New Zealand White rabbits dosed following the same protocol as the rats. Body weights of female rabbits in the mid-dose group (308 mg/m³) were higher than for the controls, but the authors did not consider this finding to be exposure-related as the high-dose group was not affected. There were several statistical differences in mean organ weights of rabbits in the low- and mid- doses, but not at the high-dose. The absolute mean kidney weights of female rabbits in the mid- and high-dose group were increased.  Female rabbits in the high-dose group (1232 mg/m³) showed 14% increased relative kidney weight compared to controls. However, the findings in the kidney weights were within the range of the historic controls, and no other kidney related parameters were affected. The authors considered the histopathological changes to be minimal spontaneous changes. No statistical differences to controls were reported from clinical chemistry or haematology. Urinalysis was not performed in the rabbits (Landry et al. 1984).

Rats, guinea pigs, rabbits and monkeys were exposed to nominal concentrations of 0 or 400 ppm (2464 mg/m³) DPGME 7 hours/day, 5 days/week for six to eight months. However, the measured vapour concentration was around 300 ppm (1848 mg/m³), as some of the substance was present as aerosols.

20 males and 20 female white rats exposed to around 1848 mg/m³ showed transient narcosis during the first weeks of exposure. No effects were seen on growth, mortality and final body weights when compared to controls. The relative liver weight was significantly increased. No report of haematological or microscopic examination of the tissues is given.

Eight male and 8 female guinea pigs exposed to around 1848 mg/m³ DPGME did not show evidence of adverse effects on gross appearance and behaviour, growth, mortality, body weights. The relative liver weights were non-significantly higher in both sexes compared to controls. Microscopic examination of the tissues revealed very slight granulation in the liver cell cytoplasm, mainly in the central area, and significantly numerous non-fatty vacuoles throughout the liver lobules.

Two male and two female rabbits and one male and one female monkey treated with DPGME at around 1848 mg/ m³ only displayed effects on the liver, namely granulation and vacuolation similar as described for the guinea pigs. (Rowe et al. 1954).

107.2.2     Oral intake

No data were found.

107.2.3     Dermal contact

The subacute percutaneous toxicity of DPGME was evaluated in groups of 8 male Porton-Wistar rats dosed 5 days/week for 4 weeks at 0, 100 and 1000 mg/kg b.w. under occluded as well as unoccluded conditions. DPGME had no effect on body weight gain nor on food intake. With respect to blood parameters, only a slightly, non-significantly elevated red blood cell count (RBC) and packed cell volume (PCV) was seen in the treated groups; however, these changes were not so apparent in the animals treated under occluded conditions. No significant changes were recorded in clinical chemistry or with respect to pathology and histopathology of several organs including the liver, bone marrow and testes. (Fairhurst et al. 1989).

Groups of 5-6 male rabbits were treated with 0, 1, 3, 5 or 10 ml/kg b.w. (corresponding to 0, 0.94, 2.8, 4.7, 9.4 g/kg b.w.) DPGME under occluded patch 5 times/week over a period of 90 days. The experiment was repeated with 0, 3 and 5 ml/kg (» 0, 2.8 and 4.7 g/kg). The mortalities recorded are listed in Table 1.

Table 1: Mortality of rabbits in 3 months dermal application

In the high dose group, 6 animals died following weight loss and narcosis. Gross examination revealed gastric retention and occasional small hemorrhagic areas in the gastric mucosa of the dead animals of this group. Occasional deaths seen at 0.94, 2.82 and 4.70 g/kg b.w. were associated with respiratory infections. There were no abnormal findings in the histological examination except the lungs, which showed pneumonia and/or empyema. Blood urea nitrogen concentrations, determined in experiment 2, were within normal limits. (Rowe et al. 1954).

107.3     Toxicity to reproduction

No specific studies on fertility were found. However, results from repeated dose studies do not indicate effects on the testes. Thus, rat testes have been reported not to be affected pathologically nor histologically following dermal exposure to 100 or 1000 mg/kg b.w., 5 days/week for 28 days (Fairhurst et al. 1962).

No changes in rat or rabbit testes weight or testicular histology were reported following exposure by inhalation to 0, 15, 50 or 200 ppm (0, 92.4, 308 or 1232 mg/m³) DPGME 6 hours/day, 5 days/week for 15 weeks (Landry et al. 1984).

107.3.1     Inhalation

Groups of 32-37 mated female Fischer 344-rats were exposed by inhalation to nominal concentrations of 0, 50, 150 or 300 ppm DPGME (0, 51, 154 or 285 ppm measured concentrations corresponding to 0, 314.2, 948.6 or 1756 mg/m³) from gestation-day 6 through 15, 6 hours/day. Clinical signs and changes in general appearance were recorded. Body weights were recorded at day 0, 6, 16 and 21. Following caesarian section, the dams were sacrificed at day 21. Liver weights were recorded. Statistically significant lower body weights at days 0, 6 and 16 in the mid-exposure group were considered insignificant as the finding was not seen at the high exposure level and was present prior to treatment. The authors evaluated that no treatment-related effects were observed in clinical signs, on feed or water consumption, on body weight or body weight gain. The following data were recorded at study termination: gross pathologic alterations in the dams, gravid uterus weight, number of foetuses in utero, number of live and dead foetuses, number of resorption sites and corporea lutea, sex and body weight of each foetus. All foetuses were also examined for soft tissue and skeletal alterations. 13 pups (4 in the control group, 2 in the low-, 4 in the mid- and 3 in the high-dose group) had malformations. The malformation types were not specified by the author. However, the numbers being low and the numbers in each of the treated groups not exceeding the controls, the authors concluded that the malformations were not due to the treatment with DPGME. (Breslin et al. 1996).

Following a similar protocol as described above for rats, groups of 16 inseminated female NZW-rabbits were exposed from day 7 through 19 of gestation, i.e. to concentrations of 0, 314.2, 949.6 or 1756 mg/m³. No treatment-related clinical signs, changes in general appearance or behaviour were observed at any exposure level. Body weights were recorded at day 0, 7, 20 and 28. One rabbit from the low-dose group was found dead on gestation day 27. A second rabbit form the low-dose group and one rabbit from the high-dose group delivered their litters on gestation day 27. Death and early deliveries were investigated and attributed to pregnancy toxaemia and not to the treatment with DPGME. The surviving dams were sacrificed on day 28 after caesarian section. No treatment-related effects on feed or water consumption or on body weights were observed. There was a large variation in body weight gains within and across groups.  However, this was considered insignificant by the authors. Liver weights were not affected by treatment. No treatment related effects on the reproduction in the dams were reported. With respect to foetal parameters, 9 foetuses (3 controls, 2 in the low-, 2 in the mid- and 2 in the high dose group) had malformations. No details on the malformation types are given. As there was no dose-response relationship and the number in each treated groups was lower than in the controls, the authors evaluated that the occurrence of malformations was not treatment related. The same evaluation was made for a statistically increased number of lumbar spurs (regarded by the authors as variations) in the low dose group (314.2 mg/m³). The authors concluded that there were no treatment-related effects on the reproductive or foetal parameters or on the incidence of malformations or variations. (Breslin et al. 1996).

107.3.2     Other routes

No studies were found.

107.4     Mutagenic and genotoxic effects

107.4.1     In vitro studies

A bacterial gene mutation assay (Ames test) conducted according to GLP in Salmonella Typhimurium strains TA1538, TA 1537, TA 1535, TA100 and TA 98 with and without metabolic activation at concentrations of 2, 10, 50, 250, 1250, and 6250 mg/plate was negative (Kirkland et al. 1983 – quoted from IUCLID 2000 and ECETOC 1995).

A cytogenetic assay conducted according to GLP using metaphase analysis of Chinese hamster ovary (CHO) cells treated with DPGME was performed with and without metabolic activation at 1.25, 2.5, 5.0 and 10 mg/ml. The test showed no differences between treated and untreated cells with or without metabolic activation. DPGME is thus not a clastogen for CHO cells. ( Kirkland 1983 – quoted from IUCLID 2000 and ECETOC 1995).

DPGME did not produce unscheduled DNA synthesis (OECD guideline 482) in a test conducted in rat hepatocytes at concentrations of 0.01, 0.00316, 0.001, 0.000316, 0.0001 and 0.0000316 M with and without metabolic activation (Mendrala et al. 1983 – quoted from IUCLID 2000 and ECETOC 1995).

107.4.2     In vivo studies

No data were found.

107.5     Carcinogenic effects

No data were found

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