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

123     Animal toxicity

123.1     Single dose toxicity

123.1.1     Inhalation

IUCLID has listed LC₅₀-values in rats ranging from above 6200 to 32500 mg/m³ for 4-hour exposure periods (IUCLID 2000).

All 6 rats exposed to 4000 ppm (16320 mg/m³) for 4 hours died. At 2000 ppm (8160 mg/m³), one out of 6 animals died. (Smyth et al. 1969 - quoted from A&H 1999).

Groups of 5 male ICR-mice were exposed to concentrated vapours of cyclohexanone, measured to 19000 mg/m³ (4750 ppm) for different periods of time (78, 90, 104 or 120 minutes). The mean time to death in mice was 100 minutes. During exposure, the animals exhibited signs of irritation, laboured respiration and CNS depression. Gross post-mortem examination showed general vascular congestion and haemorrhages of the lungs. Histopathology performed on the survivors 7 days after the 2-hour exposure revealed lung oedema and hyperplasia in the white pulp of the spleen. Findings in the brain, heart, liver adrenals, and gonads were within normal limits. (Gupta et al.1979).

In guinea pigs, concentrations of 4000 ppm (16320 mg/m³) cyclohexanone for 6 hours caused narcosis and depressed respiration. Three out of 10 animals died within 4 days of exposure. (Specht et al. 1940 - quoted from ACGIH 1991 and from A&H 1999).

123.1.2     Oral intake

LD₅₀-values in the range of 1296 to 3460 mg/kg b.w. in rats and of 1400 to 3200 mg/kg b.w. in mice have been listed in IUCLID (2000). Symptoms of acute toxicity included narcosis and laboured respiration, and the autopsy revealed peritoneal and intestinal congestion. (Gupta et al. 1979).

Minimum lethal doses for rabbits have been reported to be from 1600 up to 1900 mg/kg b.w. Sub-lethal doses of 900 to 1600 mg/kg b.w. caused narcosis, ataxia, pronounced lung oedema, and necrosis of the respiratory epithelium, heart muscle, liver and of the kidneys. (Treon et al. 1943a).

123.1.3     Dermal contact

Dermal LD₅₀-values in rabbits ranged between 794 and 3160 mg/kg b.w. (OEL 1993, A&H 1985, IUCLID 2000). However, minimum lethal doses from 10200 up to 23000 mg/kg after one hour of skin contact have also been reported. Symptoms of toxicity included severe irritation of the application site, convulsive movements, and narcosis. (Treon et al 1943a).

123.1.4     Skin irritation

Concentrations of 0, 12.4, 24.8, 49.5, and 99% cylclohexanone in cottonseed oil were applied to the shaved backs of male rabbits under occlusive patch for 24 hours. The irritation response was concentration dependent with the lowest test concentration producing a minimally detectable, transient response graded 0 on a 3-grade scale. At 24 hours after exposure, the medium test concentrations caused grade 1 and grade 2 irritation, respectively, which disappeared within 72 hours. The highest test concentration caused a grade 3 irritation lasting until day 7 after exposure. (Gupta et al. 1979).

Results in IUCLID vary from reporting cyclohexanone as being non-irritating (in an OECD guideline 404-study) to corrosive to rabbit skin. No further details of these studies are available in IUCLID, and most of the studies are not publicly available. (IUCLID 2000).

123.1.5     Eye irritation

Concentrations of 2.5, 5, 10, 20, 40, 80, or 99% cyclohexanone in cottonseed oil were instilled (volume instilled not indicated) into one eye of New Zealand White rabbits. Concentration dependent eye irritation was observed following instillation from 5% cyclohexanone, reaching a score of 3 on a 3-grade scale from 40% cyclohexanone. (Gupta et al. 1979 ).

Application of 0.02 ml of undiluted cyclohexanone to the eyes of rabbits caused severe injury of the eye with marked irritation and corneal injury. The authors graded the eye irritation as 5 on a 10-grade scale. (Carpenter et al 1946).

Seven Stauffland Albino rabbits were treated with 0.1 ml of cyclohexanone solutions in distilled water in the lower conjunctival sac of the eye. The concentrations of cyclohexanone tested were 10, 15, 25, 40, 50, 75, and 100%. Measurements of corneal thickness were performed on day 3 and scored in a 5-grade scoring system, including reversibility of effect at 24 hours and thickness ratio between treated and untreated eye. Scoring was also performed according to the EPA scoring criteria over 21 days. The irritation was dose response related, with both systems scoring the 10% solution as a mild eye irritant, and from 40% as corrosive (Morgan et al. 1987).

IUCLID (2000) reports cyclohexanone to be irritating or severely irritating to the rabbits eye.

123.1.6     Sensitisation

In a Guinea Pig Maximisation Test, groups of 25 guinea pigs were exposed to one of 2 batches of cyclohexanone resin or to cyclohexanone and challenged with 5, 10 or 20% at 24 and 48 hours. Nine, 13 and 8 animals, respectively, reacted in the re-challenge test to the first batch of cyclohexanone resin, while 2, 1 and 0, respectively, reacted to the second batch. No animals reacted at any concentration at challenge to cyclohexanone. (Bruze et al. 1988).

Another Guinea Pig Maximisation Test was performed with cyclohexanone in a validation procedure of the Mouse Ear Swelling test. The GPMT test was negative (no details on the protocol are given in the reference). The Mouse Ear Swelling Test was also negative with topical application of undiluted cyclohexanone to the left ear, while the right ear only had the vehicle applied. The swelling percentage (left ear thickness / left ear thickness × 100) was 102%. Cyclohexanone was also reported to be negative in a closed patch test in guinea pigs (no details on the protocol are given in the reference). (Gad et al. 1986).

123.2     Repeated dose toxicity

123.2.1     Inhalation

Continuous exposure of young rats to 2 ppm (8.2 mg/m³) cyclohexanone for 7 weeks and of adult rats to 8 ppm (32.6 mg/m³) for 10 weeks resulted in morphological changes in cells of the olfactory bulbs (Panhuber et al. 1987, Rehn et al. 1988 – both quoted from OEL 1993 and from A&H 1999).

Rabbits exposed to 12120 mg/m³ (corresponding to 3082 ppm), 6 hours a day, 5 days per week for 3 weeks showed weight loss, symptoms of CNS depression (narcosis, laboured breathing, ataxia, and increased salivation), and eye irritation; two of the 4 rabbits died. When exposed to lower concentrations (190, 309, 608, 773, or 1414 ppm - corresponding to 750, 1210, 2390, 3040, or 5560 mg/m³) for 10 weeks, rabbits showed evidence of eye irritation from 309 ppm (1210 mg/m³). At concentrations of 773-3082 ppm (3040-12120 mg/m³), irritation symptoms were more marked and included lachrymation, salivation and watery discharge from the nose. Narcotic effects were reported at the highest concentration. No effects were seen at any dose level on haematology. No control group was included in the study. (Treon et al. 1943b).

One rhesus monkey exposed to 2390 mg/m³ cyclohexanone (6 hours/day, 5 day/week for 10 weeks) was reported to have extensive injury in the heart muscle, the lungs, the liver, and the kidneys (not specified further). The monkey had also contracted a bronchopulmonary infection. (Treon et al. 1943b).

123.2.2     Oral intake

In a range-finding study preceding a chronic study, five F-344 rats/sex/group were given 0, 190, 400, 800, 1600, 3300, 4700, or 6500 mg/l cyclohexanone in the drinking water for 6 months (the top-dose corresponded to approximately 1000 mg/kg b.w./day). No death occurred during the study, but weight gain was depressed approximately 10% in both males and females of the highest dose group. A mild degenerative change in the thyroid gland (no details in the reference) was observed at pathological examination in 2 males administered 4700 mg/l. There were no significant histopathological changes (the changes are not specified by the authors). The authors considered a maximum tolerated concentration of 6500 mg/l for the chronic study (see 4.5.2). (Lijinsky & Kovatch 1986).   

A range-study was also performed in mice. Ten (C57BL/6 x C3H)F₁ mice/sex/group were given 0, 400, 2300, 6500, 13000, 25000, 34000, or 47000 mg/l cyclohexanone in the drinking water for 13 weeks. In the high-dose group, 1/3 of the females and 2/3 of the males died during treatment. One male of the 34000 mg/l group also died. Body weights were reduced 15% in females and 24% in males at this dose, and 19% in males of the 25000 mg/l group. At pathological examination, some of the animals of the high dose group had focal liver necrosis, and 2 high-dose females had thymus hyperplasia, whereas changes at the lower doses were minimal. A maximum tolerated concentration of 25000 mg/l for female and 13000 mg/l for male mice was considered for the chronic study (see 4.5.2). (Lijinsky & Kovatch 1986).  

123.2.3     Dermal contact

Repeated cutaneous applications of cyclohexanone (dose not specified) to 120 guinea pigs for 3-8 weeks resulted in cataract with subcapsular focal vacuolated areas in 29 animals. No effects were seen in the control animals. (Rengstorff et al. 1972 – quoted from DECOS 1993, Henschler 1994, and from A&H 1999).

In another study including 12 New Zealand White rabbits and 12 Harley albino guinea pigs of both sexes treated on a 3- inch shaved area of their backs with 0.5 ml (470 mg) undiluted cyclohexanone 3 times/week for 3 weeks, ophthalmological examination 6 months after treatment showed subcapsular vacuolisation and opacification of the lenses due to fiber degeneration both in the treated and the control group guinea pigs. No lenticular effects were recorded in the rabbits. The authors concluded that the lens changes are natural for guinea pigs thus making this species unsuited for studying this effect. (Greener and Youkilis 1984 – quoted from DECOS 1993 and from A&H 1999).

In a study in CD Sprague Dawley rats and Hartley guinea pigs treated on the back with 0.5 ml (470 mg) of undiluted or a 2% solution of cyclohexanone 3 times/week for 3 weeks, the guinea pigs of the treated as well as of the positive and the negative control groups had lenticular vacuolisation. No eye effects were seen in the rats even after prolongation of the dosing period to 13 weeks. IUCLID comments that the guinea pig is not a suitable animal model for investigation of ophthalmological effects. (Mayhew 1984 – quoted from DECOS 1993 and from IUCLID 2000).

123.2.4     Other routes

Dogs exposed by intravenous administration to 284 mg/kg b.w. cyclohexanone for 18-21 days showed lachrymation, mydriasis, salivation, ataxia, occasional convulsive movements, stupo, and/or dyspnoea/hypernoea. Absolute and relative liver weights were affected. Pathological examination of the organ showed glycogen depletion, plasma cell infiltrates round the hepatic veins, and haemosiderin deposits. Haemolysis, bone marrow hyperplasia, and extramedullary haematopoiesis were also reported. (Koeferl et al. 1981- quoted from DECOS 1993).

123.3     Toxicity to reproduction

123.3.1     Inhalation

In a two-generation study, 30 Sprague Dawley rats/sex were exposed (6hours/day, 5 days/week) to concentrations of 0, 250, 500, or 1000 ppm (0, 1020, 2040, or 4080 mg/m³) in the F₀-generation and to 0, 250, 500, or 1400 ppm (5712 mg/m³) in the F₁-generation. No adverse effects were seen in the F₀-generation. Rats in the high-dose group of the F₁-generation showed increased mortality, lachrymation, ataxia, irregular breathing, and body weight gain depression in males. The fertility of the males was reduced (no details on which parameters were examined), and the offspring of this dose group had body weight gain depression and reduced survival. No changes in reproductive organs were seen at histopathological examination of animals of the F₁ and F₂ generation. (American Biogenics Corporation 1986 – quoted from DECOS 1993, BUA 1997, and IUCLID from 2000).

CD rats (26 animals per group) were exposed by whole body inhalation to 0, 300, 650 or 1400 ppm (0, 1224, 2652 or 5712 mg/m³) cyclohexanone, 6 hours a day on gestational days 6-19. The highest concentration caused lachrymation, nasal discharge and lethargy as well as significantly reduced maternal and foetal body weights. Effects on pregnancy rate, pre-implantation loss, number of resorptions, or number of live foetuses per litter were non significant. There was a sporadic increase in skeletal and visceral variations, but no malformations were seen. The authors concluded that there was no evidence of teratogenicity. (Homan & Schroeder 1984 and Bio/dynamics 1984– quoted from DECOS 1993 and from IUCLID 2000).

Groups of 5-9 Sprague-Dawley rats were exposed to cyclohexanone at gestation days 5-20 to concentrations of 100, 250, or 500 ppm (408, 1020, or 2040 mg/m³) for 7 hours a day. Positive and negative controls were included. Only slightly lower weight gains were recorded in the dams of all treated groups. In the two highest dose-groups, a grey mottling of the lungs were observed in the dams. There was no treatment related effects on mean number of corpora lutea, implantation, foetal death, foetal weights, resorptions, or sex-ratio. There was a weak, non-significant increase in the mean percent of rudimentary ribs. In the two highest dose groups, three foetuses had heart artery malformations and 2 foetuses had skeletal malformations, but the findings were not statistically significant. The authors concluded that the substance was unlikely to be a developmental toxicant by inhalation exposure. (Samimi et al. 1989).

Mice exposed to cyclohexanone at concentrations of 300, 650 ,or 1400 ppm (1224, 2652, or 5712 mg/m³), 6 hours a day from gestation day 6 to 17 had reduction of the maternal body weight, and of the number of corpora lutea and live foetuses at the highest exposure level. There was no significant increase of external malformations. No effects were reported at lower exposure levels. (Homan & Schroeder 1984 – quoted from DECOS 1993). 

A study with CD-1 mice exposed to concentrations of 0 or 1400 ppm (5712 mg/m³) cyclohexanone, 6 hours a day from day 6 to 17 of pregnancy showed decreased body weight gain in the treated group. Clinical symptoms included lethargy, lachrymation, and white ocular discharge. The number of resorptions was significantly increased and some dams resorbed their litter totally. The number of live foetuses was significantly decreased, and there was an increase in skeletal variations, a weak significant trend of dilated renal pelvis, and one case of cleft palate. The authors concluded that 1400 ppm was toxic to the dams and the foetuses, but not teratogenic. (Biodynamics 1984 – quoted from IUCLID).

123.3.2     Oral intake

No maternal or developmental effects were seen in a study with CD-1 mice dosed with 0 or 800 mg/kg b.w./day cyclohexanone by gavage on days 8-12 of gestation, with a post-treatment period of 250 days. No effects of treatment on body weights or reproductive parameters were recorded in the treated dams. The offspring were monitored for postnatal viability, growth, morphology, locomotor activity in a figure eight maze, and reproductive function. No differences between treated and control animals were recorded for these parameters. (Gray & Kavlock 1984 – quoted from IARC 1989 and from IUCLID 2000; Gray et al 1986).

In a another study where groups of pregnant ICR/SIM were treated by gavage with 0 or 2200 mg/kg b.w. cyclohexanone from day 8-12 of gestation, 6 of 28 treated mice died while no mice of the control group died; 2 dams resorbed their litters completely. Maternal body weight gains were reduced in the treated group. Birth weights and body weight gains of the pups were depressed, but their viability was normal. (Seidenberg 1986 – quoted from IARC 1989, IUCLID 2000, and from A&H 1999).

Cyclohexanol, the major metabolite of cyclohexanone, has been reported to affect the testes of rabbits dosed with 25 mg/kg b.w./day for 40 days (Dixit et al. 1979 – quoted from OEL 1993).

123.3.3     Dermal contact

No data were found.

123.4     Mutagenic and genotoxic effects

123.4.1     In vitro studies

A number of in vitro mutagenicity assays on cyclohexanone have been performed, the studies are summarised in Table 4.

Table 4. In vitro mutagenicity studies.

123.4.2     In vivo studies

A sex-linked recessive lethal (SLRL) test, where male Drosophila melanogaster were exposed by inhalation to 50 ppm cyclohexanone for 7 hours (204 mg/m³) or to 400 ppm (1632 mg/m³) for 40 minutes, showed lethality rates of 0.25% and 1%, respectively. However, because of the non-reproducible result, the authors concluded that cyclohexanone is not mutagenic in Drosophila. (NIOSH 1980 / McGregor 1980 – quoted from DECOS 1993 and from IUCLID 2000).

Inhalation exposure of Drosophila melanogaster for 4 hours to 1900 ppm cyclohexanone did not induce gene mutation (Unpublished report from Nipro Inc. 1986 – quoted from IUCLID 2000).

Another Drosophila melanogaster gene mutation test with oral exposure to 0.1 or 100 ml/day for 3 days was negative (Goncharova 1970 citation in Chem Abstr 1972 - quoted from BUA 1993 and from IUCLID 2000).

A dominant lethal test where male rats were exposed to 50 or 400 ppm (204 or 1632 mg/m³) cyclohexanone 7 hours a day for 5 consecutive days did not induce chromosomal aberrations. Pregnancy frequency, numbers of corpora lutea and implantations sites, and survival of the embryos were not affected. (NIOSH / McGregor 1980 – quoted from DECOS 1993, BUA 1993, and from IUCLID 2000).

Cyclohexanone has been reported to induce chromosomal aberrations, including chromatid gaps, breaks, centric fusions, chromatid exchanges, and polyploidy, in bone marrow cells of rats treated subcutaneously with one or five doses of 0.1, 0.5 and 1.0 g/kg b.w. However, the test quality was criticised in BUA and in IUCLID because of lack of a control group, lack of dose-dependency, and cytotoxic effect of the doses. (De Hondt et al. 1983 - quoted from IARC 1989, DECOS 1993, BUA 1993, and from IUCLID 2000.)

In a micronucleus test in bone marrow cells of mice exposed to 204 or 1632 mg/m³ cyclohexanone in one single 7-hour-exposure or at 7 hours a day for 5 days, the frequencies of chromosomal aberrations were not increased significantly (NIOSH / McGregor 1980 – quoted from DECOS 1993, BUA 1993, and from IUCLID 2000).

123.5     Carcinogenic effects

123.5.1     Inhalation

No data were found.

123.5.2     Oral intake

In a 2-year toxicity assay, groups of 52 F344 rats/sex were exposed to 0, 3300, or 6500 mg/l in the drinking water (according to DECOS corresponding to 500, or 1000 mg/kg b.w./day). Survival was comparable in treated and control groups except for a non-significant decrease in the high-dose group. Body weight gain was significantly depressed in a dose-related way. The incidence of adrenocortical adenomas was statistically significantly increased in male rats of the low-dose group, but not at the high dose and not in females. The number of fibroadenomas in the mammary glands in females of the high dose was reduced compared to controls. The incidence of thyroid follicular-cell adenomas/carcinomas was increased with statistical significance in the high-dose group males (6/51, compared to 1/52 in controls, but this finding was not commented by the authors, thus probably not regarded as a carcinogenic effect. (Lijinsky & Kovatch 1986).

The study by Linjinsky & Kovatch included also groups of 50 or 52 (C57BL/6 x C3H)F₁ mice/sex exposed to 0, 6500, or 13000 mg/l with an additional group of 41 female mice receiving 25000 mg/l in the drinking water. Survival was depressed to 40% in females and to 70% in males at 13000 mg/l and dropped to 15% in the 25000 mg/l group of females. Body weight gains were affected resulting in 15-20% lower body weights of males at 13000 and females at 25000 mg/l from week 15 through most of the study when compared to those of the controls. Histopathology revealed hepatocellular adenomas/carcinomas in male mice in 25/51 males at 6500 mg/l and 13/46 at 13000 mg/l, while the controls had 16/52 liver tumours. Thus, no dose-relationship was seen and only the incidence of the tumours in the low-dose group was statistically significant. Alveolar-bronchiolar adenomas/carcinomas were found in male mice in a negative trend, with significance in the high dose group. The number of lymphosarcomas was statistically elevated in female mice of the 6500 mg/l group (17/50, compared to 8/52 in controls, 4/50 at 13000 mg/l and 0/41 at 25000 mg/l). The authors found this effect at only one dose level only suggestive of a weak carcinogenic effect. IUCLID finds the relevance of the finding questionable. (Lijinsky & Kovatch 1986, IUCLID 2000).

123.5.3     Dermal contact

No data were found.

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