Octamethylcyclotetrasiloxane

Toxicity to reproduction

Administrative data

Endpoint:

two-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

20.12.1996 to 09.03.2009 (experimental termination, as study completion date not given)

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with national standard methods

Data source

Reference

Reference 1

Reference Type:

study report

Title:

Unnamed

Year:

2001

Report date:

2001

Reference 2

Reference Type:

publication

Title:

A two-generation reproductive toxicity study of octamethylcyclotetrasiloxane (D4) in rats exposed by whole-body vapor inhalation.

Author:

Siddiqui, WH, DG Stump, KP Plotzke, JF Holson, and RG Meeks

Year:

2007

Bibliographic source:

Reproductive Toxicology 23: 202-215.

Reference 3

Reference Type:

other: Amendment to Report Number 2001-10000-50855

Title:

Unnamed

Year:

2009

Report date:

2009

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3800 (Reproduction and Fertility Effects)

Test guideline 2

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)

Principles of method if other than guideline:

This study evaluated the potential toxicity of whole-body vapor inhalation of octamethylcyclotetrasiloxane (D4) on reproductive capabilities in exposed F0 andF1 parental animals and the potential effects on neonatal survival, growth, and development of the F1 and F2 offspring.

Amendment to the Final Report: 2009-10000-60443.
The following changes were made:
1) A summarization of the results of the additional microscopic examinations specified in protocol amendment XIII were added to the abstract and conclusions of the report.
2) The experimental termination date was updated due to additional microscopic examinations. In addition, the experimental completion date was not presented in the original final report.
3) The results of the additional microscopic examinations specified in protocol amendment XIII were added. In addition, the morphology of the pigment was further discussed as part of the additional examinations, and it was not determined if the microscopic findings in the liver were adverse or non-adverse.
4) The additional reference was cited in the microscopic section regarding the results of the histopathologic evaluation of the liver slides.
5) Following re-examination, it was determined that no pigment was present in the liver for female no. 61179-08.
6) An additional protocol amendment was prepared following the issuance of the final report for the purpose of performing additional histopathologic evaluation of the liver slides for all animals noted with brown pigment.

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Octamethylcyclotetrasiloxane

EC Number:

209-136-7

EC Name:

Octamethylcyclotetrasiloxane

Cas Number:

556-67-2

Molecular formula:

C8H24O4Si4

IUPAC Name:

2,2,4,4,6,6,8,8-octamethyl-1,3,5,7,2,4,6,8-tetroxatetrasilocane

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, North Carolina
- Age at study initiation: (P) 16 wks; (F1, first pairing) 14-15 wks: (F1, second pairing) 26-27 wks.
- Weight at study initiation: (P) Males: 372-605 g; Females: 215-346 g; (F1, first pairing) Males: 293-619 g; Females: 199-343 g; (F1, second pairing) Males: 379-701 g; Females: 220-460 g.
- Fasting period before study: No
- Housing: Individually in suspended wire-mesh cages (after mating in plastic maternity cages with nesting material)
- Diet (e.g. ad libitum): Ad libitum (except during exposure)
- Water (e.g. ad libitum): Ad libitum (except during exposure)
- Acclimation period: at least five days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22.2 ±2.2
- Humidity (%): 40-70
- Air changes (per hr): approx. 10
- Photoperiod (hrs dark / hrs light): 12/12


IN-LIFE DATES: From: 14.01.1997 To: 25.04.2000

Administration / exposure

Route of administration:

inhalation

Type of inhalation exposure (if applicable):

whole body

Vehicle:

unchanged (no vehicle)

Details on exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Each group of animals was exposed in a 2m3 stainless steel and glass whole-body inhalation chamber.
- Source and rate of air: No data
- Method of conditioning air: No data
- Temperature, humidity, pressure in air chamber: 20-26 oC, 30-60% humidity
- Air change rate: 12-15
- Treatment of exhaust air: No data


TEST ATMOSPHERE
- Brief description of analytical method used: Gas chromatography
- Samples taken from breathing zone: no data

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: 14 days
- Proof of pregnancy: vaginal plug or sperm in vaginal smear referred to as day 0 of pregnancy
- After ... days of unsuccessful pairing replacement of first male by another male with proven fertility: No data
- Further matings after two unsuccessful attempts: No data
- After successful mating each pregnant female was caged (how): plastic maternity cages with nesting material.

F0 animals were mated once to produce the F1 generation; F1 parental animals were mated twice to produce two F2 litters. In addition, the F1 males were mated with unexposed females.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Exposure concentrations within each chamber were measured approximately every 35 minutes, yielding approximately 10 data points, during each daily exposure period by a validated gas chromatographic method.

Duration of treatment / exposure:

Exposure period: F0 and F1 males and females were exposed at least 70 days prior to mating, throughout mating, gestation (to gestation day 20), lactation, with the exception of lactation days 0-4, until euthanization.
Starting on PND 22, F1 weanlings were exposed to D4 as described for the  F0 generation.
The F2 pups were not directly exposed to D4.
Duration of test: ca. 39 months.

Frequency of treatment:

6 hr/day, 7 days/week

Details on study schedule:

- Selection of parents from F1 generation when pups were 4 days of age.
- Age at mating of the mated animals in the study: F1 first mating: 14-15 wks; F1 second mating: 26-27 wks; F1 treated males with untreated females: Males: 28-29 wks and females 12 wks.
- 30/pups/sex were selected on PND 28 to comprise the F1 generation.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

70 ppm (nominal)

Doses / concentrations 2

Dose / conc.:

300 ppm (nominal)

Doses / concentrations 3

Dose / conc.:

500 ppm (nominal)

Doses / concentrations 4

Dose / conc.:

700 ppm (nominal)

Doses / concentrations 5

Dose / conc.:

71 ppm (analytical)

Remarks:

F0 generation

Doses / concentrations 6

Dose / conc.:

298 ppm (analytical)

Remarks:

F0 generation

Doses / concentrations 7

Dose / conc.:

502 ppm (analytical)

Remarks:

F0 generation

Doses / concentrations 8

Dose / conc.:

700 ppm (analytical)

Remarks:

F0 generation

Doses / concentrations 9

Dose / conc.:

71 ppm (analytical)

Remarks:

F1 generation

Doses / concentrations 10

Dose / conc.:

301 ppm (analytical)

Remarks:

F1 generation

Doses / concentrations 11

Dose / conc.:

502 ppm (analytical)

Remarks:

F1 generation

Doses / concentrations 12

Dose / conc.:

702 ppm (analytical)

Remarks:

F1 generation

No. of animals per sex per dose:

30/sex/group

Control animals:

other: yes, exposed to filtered air

Details on study design:

- Dose selection rationale: Based on previous reproductive toxicity studies.

Positive control:

None

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily for appearance, behaviour, moribundity and mortality.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Weekly

BODY WEIGHT: Yes
- Time schedule for examinations: F0 and F1 males: weekly beginning with initiation of exposure and continuing until sacrifice. F0 and F1 females: weekly beginning at initiation of exposure and continuing until evidence of copulation. Then on gestation days 0, 7, 10, 14 and 20, and on lactation days 1, 4, 7, 14 and 21. After weaning (lactation day 21), weekly body weights were recorded for F0 females until scheduled necropsy. For the F1 females body weights were recorded beginning after weaning of the F2a litters on lactation day 21 and continued until evidence of copulation was observed after the second pairing. Once evidence of mating was observed, F1 body weights were recorded on gestation days 0, 7, 10, 14 and 20, and on lactation days 1 and 4.

FOOD CONSUMPTION: Yes, on a weekly basis, but during the mating period.
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

FUNCTIONAL OBSERVATION BATTERY: Yes, of all F1 female rats on gestation day 10 and lactation day 20.

reproductive parameters (days between pairing and coitus, fertility, fecundity, length of gestation, pup growth and survival, testicular and epididymal sperm count, 
sperm production rate, sperm motility and morphology, ovarian primordial follicle count), developmental landmarks (e.g., balanopreputial separation and vaginal patency), developmental neuropathology, selected organ weights, gross pathology, histopathology.

NEUROPATHY: yes

Oestrous cyclicity (parental animals):

Vaginal smears were prepared daily to determine the stage of the oestrus cycle for each F0 and selected F1 females, beginning 21 days prior to pairing and continuing until evidence of copulation was observed. Following weaning of the F2a litters and a rest period of at least 16 days, daily vaginal smears were performed for each F1 female for 15 days prior to the second pairing with the F1 males.

Sperm parameters (parental animals):

Parameters examined in [all/P/F1/F2] male parental generations: epididymal weights, testicular and epididymal sperm count, sperm production rate, sperm motility and morphology.

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 8 pups/litter (4/sex/litter as nearly as possible); excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in [F1 / F2 / F3] offspring: pup appearance and behaviour, pup growth and survival, developmental landmarks (anogenital distance on PND 1 for all F1 and F2a pups, balanopreputial separation in F1 and F2a males, vaginal patency in F1 and F2a females), sex ratio, developmental neuropathology and neurobehavioral evaluations.


GROSS EXAMINATION OF DEAD PUPS: yes, for external and internal abnormalities; possible cause of death was not determined for pups born or found dead.]

Postmortem examinations (parental animals):

SACRIFICE
All surviving animals. F0 adults: following the selection of the F1 generation and completion of detailed clinical obs; F1 males: following completion of breeding with unexposed females; F1 females: on lactation day 4, on post-mating day 25, or 25 days following completion of the second breeding period.

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.

HISTOPATHOLOGY / ORGAN WEIGHTS (control and high concentration groups, as well as animals from F0 and F1 parental animals that died spontaneously or in extremis: Organs examined at necropsy (macroscopic and microscopic): Complete gross necropsy was conducted. Organ weights:  Adrenals, prostate, brain, seminal vesicles with epididymides (total and cauda), coagulating glands, heart, kidneys, spleen, liver, testes, lungs, thymus, ovaries, thyroid, pituitary, and uterus.
Histopathologic evaluation: complete histopathologic evaluation of tissues and organs including  adrenal glands, brain, cervix, coagulating gland, epididymis (right caput, corpus, and cauda), kidneys, liver, lungs, ovaries, penis, pituitary gland, prepuce, preputial gland, prostate, seminal vesicles, testis (right), thyroid, uterus, vagina, and vas deferens, all gross (internal) lesions.

Postmortem examinations (offspring):

SACRIFICE
Prior to the weaning of the F1 pups, 30 pups/sex/group were randomly selected for the F1 parental generation and for evaluation of developmental landmarks. All surviving non-selected F1 weanlings were sacrificed on PND 21 or 28 and necropsied with an emphasis on developmental morphology evaluations.
Similarly, prior to weaning of the F2a pups, 30 pups/sex/group were randomly selected for neurobehavioural testing, developmental landmarks, and/or neuropathology and brain weight/dimension measurements. In addition, 10 F2 pups/sex/group were selected for brain weight measurements and/or neuropathology on PND 11. All remaining F2a weanlings were sacrificed and necropsied on PND 21, with an emphasis placed on developmental morphology evaluations. F2a pups not allocated for neuropathology and brain examinations received a complete necropsy on PND 70.
F2b and F2c litters were sacrificed and discarded on PND 4.


GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.


HISTOPATHOLOGY / ORGAN WEIGHTS: The brains, spleens and thymus glands from all unselected F1 and F2a weanlings necropsied on PND 21 or 28 were weighed. Additional microscopic examinations (Birefringence of pigment under polarized light) were performed for F1.

Statistics:

Statistical methods:
Chi-Square test with Yates' correction factor for parental mating and fertility indices
One-way ANOVA with Dunnett's test for the following endpoints:  Pre-coital intervals, Parental weekly,
gestational and lactational body weight and food consumption data, Gestation lengths, Sperm numbers, 
Sperm production rates, Organ weights*, Ovarian primordial follicle counts, Numbers of pups born**, 
Live litter sizes**, Anogenital distances**, Pup body weights**, Continuous FOB data, Startle response data, 
Balanopreputial separation, Vaginal patency
Fisher's Exact Test - Scalar/descriptive FOB data Two-way repeated measures ANOVA - Motor activity data
Kruskal-Wallis test with Mann-Whitney U test - Sperm motility, Sperm morphology, Pup sexes at birth
(% males per litter)**, Postnatal survival**
Kolmogorov-Smimov test (one-tailed test) - Histopathological findings * for PND 21 organ weights, 
the litter was the experimental unit of evaluation **litter used as experimental unit

Reproductive indices:

Mating and fertility indices: male and female mating index; male and female fertility index;

Offspring viability indices:

Live litter size, postnatal survival between birth and PND 0 or PND 4, postnatal survival for all other intervals,

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Other effects:

not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

effects observed, treatment-related

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS): F0: In the control, 500 and 700 ppm groups, 1/30, 1/30 and 1/30 males and 0/30, 0/30 and 4/30 females, respectively, were found dead between weeks 1 and 16. An additional 1/30 males in the 700 ppm group was authanised in extremis on day 17. Two females of the 700 ppm group died of dystocia on days 96 and 94. The other two females in the 700 ppm group died on gestation day 22 and study day 117. Liver necrosis played a part in one death, and the other was due to kidney failure. Ejaculatory plugs were noted in an exposure-related manner in most of the males throughout the exposure. The significance of this finding is unknown. No other significant clinical signs were noted at any D4 concentration. 
F1: 1/30 and 1/30 females died in 500 and 700 ppm groups between weeks 19 and 43. There were no clinical signs in animals that survived to scheduled necropsy.

BODY WEIGHT: during the first week of exposure, statistically significant reductions in mean body weight gain were observed in males and females in the 700 ppm group and in females in the 500 ppm group in the F0 animals only. Mean body weight gain was reduced (statistically significant) during gestation in the 700 ppm group in both the F0 and F1 parental animals.

FOOD CONSUMPTION: Weekly food consumption was reduced during week 0-1 in the 700 ppm group males, consistent with the reduced body weight gain in this group. Food efficiency was reduced in the 700 ppm group during gestation days 14-20 and the overall gestation period. This finding was consistent with the reduced body weight gain observed in this group at the end of the gestation period.

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS): Microscopic evaluation of the ovaries, uterus, vagina, mammary gland and pituitary gland from the 0, 70, 300, 500, and 700 ppm F1 females suggested a subtle non-exposure responsive effect, characterized by perturbation of the estrous cycle and accelerated reproductive senescence in the F1 (but not F0) females at 70, 300, and 500 ppm, with a more obvious effect at 700 ppm.

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS): No adverse effects were observed on male functional reproductive parameters, on male spermatogenic endpoints, on microscopic evaluation of male reproductive tissue, or when the D4-exposed F1 males were mated with the unexposed, nulliparous females, demonstrating that the reproductive toxicity observed was due to D4 exposure of the female.


REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS): Extended parturition and/or dystocia were observed in two and three F0 females in the 500 and 700 ppm groups, respectively, and in one F1 dam each in the 300, 500, and 700 ppm groups. Two of the three F0 700 ppm group dams and the one F1 500 ppm group female died as a result of the dystocia. Fertility indices (OECD and EPA methods of calculation) were statistically significantly reduced in the 700 ppm group for the first F1 mating period. In the second F1 mating period, male and female fertility indices were statistically significantly reduced in the 500 and 700 groups (EPA method). The male and female fertility indices for the second F1 mating were also reduced in a non-exposure responsive manner in the 70 and 300 ppm groups using both methods of calculation, although the differences from the control group were not statistically significant.

ORGAN WEIGHTS (PARENTAL ANIMALS): Statistically significant increases in mean kidney weights (absolute and relative to final body weight and relative to brain weight) were observed in males in the 500 and 700 ppm groups. A statistically significant increase (relative to body weight) was also observed in the 300 ppm males. Increased mean liver weights (statistically significant; absolute and relative to final body weight and relative to brain weight) were observed in females in the 300, 500 and 700 ppm groups and in males of the 700 ppm group.

GROSS PATHOLOGY (PARENTAL ANIMALS): No adverse findings at scheduled sacrifice.

HISTOPATHOLOGY (PARENTAL ANIMALS): For scheduled deaths: The incidence of tubular mineral mineralisation in the kidneys of the 500 and 700 ppm group males (6/29 and 12/28 males) was increase relative to controls (0/29 males). The severity of the effect was minimal in the 500 ppm and moderate in the 700 ppm group. This mineralisation was considered to be exposure-related, but was not adverse. The incidence of centrilobular hepatocyte hypertrophy was increased in females in the 500 and 700 ppm groups (8/30 and 6/26) relative to controls (1/30). This finding was considered to be exposure-related, but not adverse.
Increased alveolar histiocytosis was observed in males of the 500 and 700 ppm groups and females in all exposure groups. This effect was attributed to treatment. However, since the effect is an incidental finding in older rats, the increase was considered to represent D4-related exacerbations of spontaneous lesions.
Examinations of the ovaries, uterus, vagina, mammary gland and pituitary gland and the establishment of the phase of the oestrus cycle for each female in the control and 700 ppm group revealed no exposure-related effects.

FUNCTIONAL OBSERVATION BATTERY: No adverse treatment-related findings.

Effect levels (P0)

Key result

Dose descriptor:

NOAEC

Effect level:

300 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Based on reductions in body weight gains.

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Mortality / viability:

mortality observed, treatment-related

Body weight and weight changes:

no effects observed

Sexual maturation:

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

no effects observed

Details on results (F1)

VIABILITY (OFFSPRING): Statistically significant reductions in mean live litter sizes and mean number of pups born were observed in the 500 and 700 ppm groups for the F0 animals, and statistically significant reductions were noted for the first mating period in the F1 animals for the mean live litter size in the 500 and 700 ppm groups and for mean number of pups born in the 700 ppm group. Slight decreases (not statistically significant) were also observed in both of these parameters in the 300 ppm group for the F0 generation. In the F1 generation, reductions (not statistically significant) in mean numbers of pups born were observed in the 500 ppm group for the first mating and the 700 ppm group for the second mating. In addition, mean live litter sizes were slightly reduced (not statistically significant) in the 70 and 300 ppm groups for the first mating and in all groups for the second mating. The reductions following the second mating period occurred in a non-exposure responsive manner in the 70, 300 and 500 ppm groups.
When the F1 males were paired with unexposed females, no effects on reproductive performance were observed. In the F1 generation, mating indices were reduced in the 700 ppm group for the first and second matings (statistically significant for the females in both matings and for males in the second mating).

CLINICAL SIGNS (OFFSPRING): No adverse effects.

BODY WEIGHT (OFFSPRING): Mean F1 pup weights were increased in the 700 ppm group compared with the controls on PND 1 and 4. These increases were considered to be related to the reduced mean live litter size in this group. Mean pup body weights were not significantly affected by exposure on PND 7, 14, 21 and 28.

SEXUAL MATURATION (OFFSPRING): No adverse effects were observed at any exposure level on anogenital distance, vaginal patency, and preputial separation.

ORGAN WEIGHTS (OFFSPRING): Mean absolute and relative organ weights (brain, spleen and thymus) of the unselected F1 weanlings euthanised on PND 21 were comparable to the control group values. At the PND 28 necropsy of unselected weanlings, mean spleen weights were reduced in males in the 300, 500 and 700 pp groups and in females of the 500 and 700 ppm groups. These decreases were generally statistically significant and could have been related to exposure to D4.

GROSS PATHOLOGY (OFFSPRING): No internal findings that could be attributed to parental exposure to D4 were noted at the necropsies of pups that were found dead prior to weaning or PND 22 and 28. At scheduled necropsy of unselected F1 weanlings on PND 21 and 28, there were no exposure-related findings.

HISTOPATHOLOGY (OFFSPRING): Brown pigment in the bile duct areas of the liver was observed in an exposure-related manner in the F1 generation males and females in the 300, 500 and 700 ppm groups. Under polarised light some pigment accumulations show birefringence, but this finding was not consistent in size or between animals, and this birefringence often showed in only parts of the pigment deposits.

OTHER FINDINGS (OFFSPRING): No developmental neurotoxicity was observed in the F2a generation.

Effect levels (F1)

Key result

Dose descriptor:

NOAEC

Remarks:

Reproductive effects

Generation:

F1

Effect level:

300 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Based on reduced fertility indices and reduced mean live litter sizes.

Overall reproductive toxicity

Reproductive effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

In a guideline, GLP, inhalation two-generation reproductive toxicity study in rats (reliability score 1), statistically significant reductions in mean live litter sizes and mean number of pups born were observed in the 500 and 700 ppm D4 groups for the F0 animals, and statistically significant reductions were noted for the first mating period in the F1 animals for the mean live litter size in the 500 and 700 ppm groups and for mean number of pups born in the 700 ppm group. When the F1 males were paired with unexposed females, no effects on reproductive performance were observed. In the F1 generation, mating indices were reduced in the 700 ppm group for the first and second matings (statistically significant for the females in both matings and for males in the second mating). Fertility indices were statistically significantly reduced in the 700 ppm group for the first F1 mating period. In the second F1 mating period, male and female fertility indices were statistically significantly reduced in the 500 and 700 ppm groups. The male and female fertility indices for the second F1 mating were also reduced in a non-exposure responsive manner in the 70 and 300 ppm groups, although the differences from the control group were not statistically significant. Microscopic evaluation of the ovaries, uterus, vagina, mammary gland and pituitary gland from the 0, 70, 300, 500, and 700 ppm F1 females suggested a subtle non-exposure responsive effect characterized by perturbation of the estrous cycle and accelerated reproductive senescence in F1 (but not F0) females at 70, 300, and 500 ppm, with a more obvious effect at 700 ppm.

In addition, some hepatic brown pigment accumulation in the bile duct was observed in the F1 generation males and females in the 300, 500 and 700 ppm groups. Under polarised light some pigment accumulations show birefringence, but this finding was not consistent in size or between animals, and this birefringence often showed in only parts of the pigment deposits.

The NOAEC for reproductive toxicity and general systemic toxicity was therefore 300 ppm.