Sodium p-chloro-m-cresolate

Toxicity to reproduction

Administrative data

Endpoint:

two-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

13 Jun 2005 - 31 May 2006

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2006

Report date:

2006

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)

Version / remarks:

adopted in 2001

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3800 (Reproduction and Fertility Effects)

Version / remarks:

adopted in 1998

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: M.A.F.F. in Japan 12 Nousan No 8147

Version / remarks:

adopted in 2000

GLP compliance:

yes (incl. QA statement)

Remarks:

Ministerium für Umwelt und Naturschutz, Landwirtschaft und Verbraucherschutz des Landes Nordrhein-Westfalen, Düsseldorf, Germany

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Chlorocresol

EC Number:

200-431-6

EC Name:

Chlorocresol

Cas Number:

59-50-7

Molecular formula:

C7H7ClO

IUPAC Name:

4-chloro-3-methylphenol

Test material form:

solid

Details on test material:

Batch No.: CHT0126 and CHA0152

Test animals

Species:

rat

Strain:

other: Wistar Crl: (WI) WU BR

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River GmbH, Sulzfeld, Germany
- Females nulliparous and non-pregnant: yes
- Age at study initiation: (P) 5 - 6 wks; (F1) 4 wks
- Weight at study initiation: (P) Males: 113 - 149 g; Females: 95 - 122 g; (F1) Males: 61 - 120 g; Females: 62 - 110 g
- Housing: individually except when co-housed for matings or with litters in Makrolon® cages Type IIIh on low-dust soft-wood shavings or nesting material
- Diet: Kliba 3883.9.25 (Provimi Kliba SA, Kaiseraugst, Switzerland), ad libitum
- Water: tap water, ad libitum
- Acclimation period: about one week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23 ± 2
- Humidity (%): 55 ± 5
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12 / 12

Administration / exposure

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

DIET PREPARATION
- Rate of preparation of diet (frequency): at least weekly
- Mixing appropriate amounts with (Type of food): Kliba 3883.9.25

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: over night at a maximum of 12 times during the three-week mating period until proof of pregnancy
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
- Females found sperm-positive after the first mating day but not shown to be pregnant (lack of weight gain within 14 days following insemination) were co-housed again over one week with the same male without checking insemination or measuring body weight and food intake during possible further pregnancy.
- After successful mating each pregnant female was caged: individually

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Checks on homogeneity (low and high concentration only) and contents of active ingredient (all doses including 0 ppm) were done at several times during the study using reversed phase high-performance liquid chromatography (HPLC) with UV detection. Homogenous distribution was proven in samples of 100 and 12000 ppm formulations taken from the top, middle and bottom of the mixture. Stability analysis of the same samples revealed that test substance concentration was within the range of ± 20% of the initially measured concentrations after storage for up to 15 days at animal room temperature (101 and 107%, respectively) as well as after freezer storage for up to 15 days (104 and 112%, respectively). Additionally, the absence of the test substance in the control diet was confirmed. Concentration analysis was conducted at six time points during the study. The test substance concentration was in the range of ± 20%.

Duration of treatment / exposure:

from beginning of the study until sacrifice (P and F1 generations; males: about 10 weeks before pairing until termination; females: about 10 weeks before pairing, throughout pairing, gestation and lactation)
approximately 4 weeks (F2a and F2b generation; from weaning until termination on Day 28 - 30 of age)

Frequency of treatment:

continously (via diet)

Details on study schedule:

- Selection of parents from F1 generation when pups were 28 days of age.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

750 ppm

Remarks:

during premating period (about 10 weeks):
equivalent to 63.8 and 80.1 mg/kg bw/day in males and females of the P0 generation, respectively and equivalent to 74.5 and 90.4 mg/kg bw/day in males and females of the P1 generation, respectively

Doses / concentrations 2

Dose / conc.:

3 000 ppm

Remarks:

during premating period (about 10 weeks):
equivalent to 247.8 and 298.2 mg/kg bw/day in males and females of the P0 generation, respectively and equivalent to 288.4 and 364.5 mg/kg bw/day in males and females of the P1 generation, respectively

Doses / concentrations 3

Dose / conc.:

12 000 ppm

Remarks:

during premating period (about 10 weeks):
equivalent to 1043.0 and 1189.7 mg/kg bw/day in males and females of the P0 generation, respectively and equivalent to 1204.9 and 1263.4 mg/kg bw/day in males and females of the P1 generation, respectively

No. of animals per sex per dose:

25

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: Dose selection was based on results of an one-generation pilot study where rats received 0, 750, 3000 and 12000 ppm of the test substance. In this study 750 and 3000 ppm were tolerated without toxicologically relevant changes and without effects on reproduction parameters. At 12000 ppm terminal body weight was depressed in males and females of the P generation and terminal pup weight depression was noted in males and females of the F1 generation. The reproduction was affected at 12000 ppm as well. Thus, due to systemic and reproductive toxicity at 12000 ppm, this 2-generation reproduction study was dosed at 0, 750, 3000 and 12000 ppm.

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily (once daily on weekends and public holidays)
- Cage side observations included: mortality, morbidity, signs of illness, clinical reactions to treatment, general state of health, behavior, condition of the fur and the orifices, excretory products, prolonged parturition

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: prior to the first administration and weekly thereafter as well as on Days 0, 7, 14 and 20 during pregnancy and on Days 0, 7, 14, 21 and 28 during lactation

BODY WEIGHT: Yes
- Time schedule for examinations: directly prior to the first adrninistration and weekly thereafter weekly up to necropsy as well as on Days 0, 7, 14 and 20 during pregnancy and on Days 0, 7, 14, 21 and 28 during lactation

FOOD CONSUMPTION AND COMPOUND INTAKE:
The individual food consumption was measured by weighing the quantity of food provided and back-weighing the amount, which remained unconsumed as follows: weekly from week 1 up to necropsy (males; except during mating period); weekly from week 1 up to mating (females), during pregnancy Day 0-7, 7-14, 14-20 (females), during lactation Day 0-4, 4-7 (females). From these food intake data the intake of the test compound was calculated per animal and/or per kg body weight per day, week or for a given period.

WATER CONSUMPTION: No

Oestrous cyclicity (parental animals):

Vaginal smears were taken daily over 19 consecutive days prior to the mating period using a flame-sterilized platinum loop. The smears were plated out on slides and stained for about 1 min with MAY GRÜNWALD´S Eosine-methylene blue solution modified for microscopy. The smears were examined microscopically for large serrated cells indicating that estrus had occurred.

Sperm parameters (parental animals):

Parameters examined in all surviving P male parental generations of the control and 12000 ppm group:
sperm motility, sperm morphology, sperm count in epididymides, other: spermatid head density in the testis

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 examined for external defects, killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in F1 and F2 offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, anogenital distance (AGD; in F2a pups only)

GROSS EXAMINATION OF DEAD PUPS:
yes, for external abnormalities; possible cause of death was not determined for pups born or found dead. lung flotation in water was performed to determine to determine whether it was a live or dead born.

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals when no longer needed for matings.
- Maternal animals: All surviving animals at weaning of 28 days old pups or up to 2 days later.

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

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated in Table 1 were prepared for microscopic examination and weighed, respectively.

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring not selected as parental animals and all F2 offspring were sacrificed at 28 days of age.
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows:

GROSS NECROPSY
- Gross necropsy consisted of macroscopical investigation with particular attention on the organs of reproduction including visible skeletal abnormalities as far as possible.

HISTOPATHOLOGY / ORGAN WEIGTHS
Brain, spleen, thymus, uterus and kidneys of one male and one female out of the firstly necropsied 5 litters per group were fixed in 10% formalin.Grossly abnormal tissues if any were fixed in all pups/weanlings. The brain, spleen, thymus and uterus of one male and one female per litter were trimmed and weighed as soon as possible after dissection. The ratio of organ weights to body weights was calculated. Therefore, all these weanlings were weighed at day of necropsy.

Statistics:

Analysis of Variance (ANOVA) and in case of significant results Dunnett's test as post hoc test (body weights, body weight gains, food consumption, number of implantation sites per female, number of viable pups per female, organ weights, data on estrus determinations, time to insemination, life birth, viability and lactation rate); 2 by N CHI² test in case of significant differences Fisher's exact test with Bonferroni correction (number of viable pups per group based on the number of implantations, insemination, fertility, gestation and rearing index); Kruskall-Wallis test and in case of significant differences Dunnett's test (number of prenatal loss per litter); Wilcoxon Mann-Whitney U test (ovarian follicle counts); statistical significance at p ≤ 0.05.

Reproductive indices:

Insemination index (%) = (No. of sperm positive females* / No. of females co-housed with a male) x 100
Fertility index (%) = (No. of pregnant females / No. of sperm positive females*) x 100
Gestation index (%) = (No. of females completing delivery / No. of pregnant females) x 100
Rearing index (%) = (No. of females rearing a litter up to Day 21 postpartum / No. of females that delivered a litter) x 100

* including pregnant females ( showing implantations ), which were not sperm positive

Offspring viability indices:

Live birth index* (%) = (No. of live pups at birth / total No. of pups born) x 100
Viability index* (%) = (No. of live pups on Day 4 pre-culling / No. of live pups born) x 100
Lactation index* (%) = (No. of live pups after three weeks / No. of live pups after four days (after culling)**) x 100

* index calculation from litter means
** moribund pups that died during the course of culling were not included

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

12000 ppm: 1/25 female was sacrificed pre-scheduled due to poor general condition after prolonged parturition (incidental)

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

12000 ppm: statistically significantly decreased mean body weights and body weight gain in males and females during pre-mating; statistically significantly decreased mean body weights and body weight gain in females during gestation; statistically significantly decreased mean body weights in females during lactation

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Endocrine findings:

not specified

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

MALES
12000 ppm: increased observation of periportal cytoplasmic change in the liver often coincided with reduced hepatocellular glycogen storage and reduced periportal fat storage in favour of a more diffuse pattem; increased simple dilation of the papillary tubules in the kidney; decreased secondary changes belonging to chronic progressive nephropathy (hyaline casts/dilated tubules)

FEMALES
12000 ppm: adaptive periportal hypertrophy/eosinophilia in the liver and reduced hepatocellular glycogen storage; brownish inclusions in the proximal tubules (proved as lipofuscin accumulations) and dilated cortico-medullary tubules in the kidney; increased secondary changes belonging to chronic progressive nephropathy (basophilic tubules); atrophy of the ovaries; increased metestrus and decreased diestrus; atrophy of the vaginal epithelium

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

effects observed, non-treatment-related

Description (incidence and severity):

Control: 1/25 spermless male

Reproductive performance:

no effects observed

Details on results (P0)

ORGAN WEIGHTS
Effects on the weights of kidneys and spleen in females, liver in males, prostate, seminal vesicles and uterus as well as increased relative weights of the brain and testes are considered to be due to differences in body weights, because relative weights were not altered statistically significantly or were statistically significantly increased. Statistically significantly changed absolute and relative adrenal weights (partly in all male dose groups) are not attributed to the treatment with the test substance as dose dependencies are absent. This is true also for the elevated absolute pituitary weights noted for 3000 ppm rats. The increased relative thyroid weights of 3000 and 12000 ppm females are interpreted to be not attributable to the treatment with the test substance as the deviations to the control value were too small.

Effect levels (P0)

1

Dose descriptor:

NOAEL

Remarks:

systemic toxicity

Effect level:

3 000 ppm

Based on:

test mat.

Remarks:

equivalent to 247.8 and 298.2 mg/kg bw/day for males and females, respectively

Sex:

male/female

Basis for effect level:

body weight and weight gain

organ weights and organ / body weight ratios

histopathology: non-neoplastic

2

Dose descriptor:

NOAEL

Remarks:

reproductive toxicity

Effect level:

>= 12 000 ppm

Based on:

test mat.

Remarks:

equivalent to 1043.0 and 1189.7 mg/kg bw/day for males and females, respectively

Sex:

male/female

Basis for effect level:

other: highest dose tested

Target system / organ toxicity (P0)

Critical effects observed:

no

Results: P1 (second parental generation)

General toxicity (P1)

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

750 ppm: 1/25 male was sacrificed after one week of co-housing period due to clinical emaciation, high-stepping gait, piloerection, gnawed fur and food spillage

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

3000 ppm: statistically significantly decreased mean body weight gain in females during lactation (females generating the F2b generation only)
12000 ppm: statistically significantly decreased mean body weights and body weight gain in males and females during pre-mating; statistically significantly decreased mean body weights in females during gestation; statistically significantly decreased mean body weights and body weight gain in females during lactation

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

3000 ppm: statistically significantly decreased food consumption in females during lactation
12000 ppm: decreased food consumption in females during pre-mating (about 18%; not statistically significant)

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

effects observed, non-treatment-related

Description (incidence and severity):

12000 ppm: increased water intake (qualitatively determination) during lactation in 2/25 females

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Endocrine findings:

not specified

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

MALES
750 ppm: statistically significantly increased absolute and relative (to body weight) pituitary weight (non-treatment-related); statistically significantly increased absolute brain weight (non-treatment-related)
3000 ppm: statistically significantly decreased absolute and relative (to body weight) liver weight
12000 ppm: statistically significantly decreased absolute kidney and spleen weight (non-treatment-related); statistically significantly decreased absolute and relative (to body weight) liver weight; statistically significantly increased absolute and relative (to body weight) seminal vesicles weight; statistically significantly increased brain and testes weight relative to body weight (non-treatment-related)

FEMALES
12000 ppm: statistically significantly increased kidney, liver and spleen weight relative to body weight; statistically significantly decreased absolute ovaries weight; statistically significantly decreased absolute uterus weight (non-treatment-related); statistically significantly decreased absolute brain weight but statistically significantly increased brain weight relative to body weight (non-treatment-related)

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

FEMALES
12000 ppm: emaciation in 7/25 females; dilated stomach and cecum in 3/25 and 4/25 females, respectively

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

MALES
12000 ppm: increased observation of periportal cytoplasmic change in the liver often coincided with reduced hepatocellular glycogen storage and reduced periportal fat storage in favor of a more diffuse pattem; papillary necrosis in the kidney; increased simple dilation of the papillary tubules in the kidney; decreased secondary changes belonging to chronic progressive nephropathy (hyaline casts/dilated tubules, basophilic tubules)

FEMALES
3000 ppm: adaptive periportal hypertrophy/eosinophilia in the liver; dilated corticomedullary tubules in the kidney in a smal number of animals
12000 ppm: adaptive periportal hypertrophy/eosinophilia in the liver and reduced hepatocellular glycogen storage; brownish inclusions in the proximal tubules (proved as lipofuscin accumulations) and dilated cortico-medullary tubules in the kidney; atrophy of the ovaries; increased metestrus and decreased diestrus; atrophy of the vaginal epithelium; statistically significant decrease in the number of growing follicles and corpora lutea

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Details on results:

ORGAN WEIGHTS
Effects on the weights of kidneys, spleen, brain and testes in males as well as uterus and brain in females are considered to be due to differences in body weights, because relative weights were not altered statistically significantly or were statistically significantly increased.

Reproductive function / performance (P1)

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

effects observed, non-treatment-related

Description (incidence and severity):

Control: 2/25 spermless males

Reproductive performance:

no effects observed

Effect levels (P1)

1

Dose descriptor:

NOAEL

Remarks:

systemic toxicity

Effect level:

750 ppm

Based on:

test mat.

Remarks:

equivalent to 74.5 and 90.4 mg/kg bw/day for males and females, respectively

Sex:

male/female

Basis for effect level:

body weight and weight gain

organ weights and organ / body weight ratios

histopathology: non-neoplastic

2

Dose descriptor:

NOAEL

Effect level:

>= 12 000 ppm

Based on:

test mat.

Remarks:

equivalent to 1204.9 and 1263.4 mg/kg bw/day for males and females, respectively

Sex:

male/female

Basis for effect level:

other: highest dose tested

Target system / organ toxicity (P1)

Critical effects observed:

no

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

Viability indices were not affected in F1 pups.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

12000 ppm: decreased litter weights from Day 14 onwards (12 - 18%); statistically significantly decreased pup weights from Day 14 onwards

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Food consumption during the 10 week pre-mating period of the F1 generation becoming P1 generation is shown in section "Results: P1 generation".

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

effects observed, treatment-related

Description (incidence and severity):

3000 ppm: statistically significantly increased age for vaginal opening (non-treatment-related)
12000 ppm: statistically significantly decreased balano-preputial separation at mean body weight; statistically significantly increased age for vaginal opening

Anogenital distance (AGD):

not examined

Nipple retention in male pups:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

MALES
12000 ppm: statistically significantly decreased absolute brain weight but statistically significantly increased brain weight relative to body weight; statistically significantly decreased absolute and relative (to body weight) spleen weight

FEMALES
12000 ppm: statistically significantly decreased absolute brain weight but statistically significantly increased brain weight relative to body weight; statistically significantly decreased absolute and relative (to body weight) spleen weight; statistically significantly decreased absolute thymus weight

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

Other effects:

no effects observed

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Details on results (F1)

SEXUAL MATURATION
The statistically significantly increased age for vaginal opening at 3000 ppm was not considered treatment-related as the values lay within the range of historical controls. Effects on the balano-preputial separation and vaginal opening at 12000 ppm were associated with reduced body weight data and, thus, considered as a consequence of delayed general development.

ORGAN WEIGHTS
Reduced absolute and (mostly) increased relative brain weights observed in male and female pups at 12000 ppm were considered to be secondary to depressed pup weights.

Effect levels (F1)

1

Dose descriptor:

NOAEL

Remarks:

systemic toxicity

Generation:

F1

Effect level:

3 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

organ weights and organ / body weight ratios

2

Dose descriptor:

NOAEL

Remarks:

developmental toxicity

Generation:

F1

Effect level:

3 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

sexual maturation

Target system / organ toxicity (F1)

Critical effects observed:

no

Results: F2 generation

General toxicity (F2)

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

F2a pups: relatively low viability indices at 0 and 3000 ppm compared to those at 750 or 12000 ppm
F2b pups: relatively low viability indices at 0 and 3000 ppm compared to those at 750 or 12000 ppm

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

F2a pups:
12000 ppm: decreased litter weights from Day 14 onwards (11 - 17%); statistically significantly decreased pup weights from Day 14 onwards

F2b pups:
3000 ppm: statistically significantly decreased pup weights from Day 14 onwards (females)
12000 ppm: decreased litter weights from Day 14 onwards (11 - 17%); statistically significantly decreased pup weights from Day 14 onwards (females) and from Day 21 onwards (males)

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Anogenital distance (AGD):

no effects observed

Nipple retention in male pups:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

F2a - MALES
12000 ppm: statistically significantly decreased absolute brain weight but statistically significantly increased brain weight relative to body weight; statistically significantly decreased absolute spleen and thymus weight

F2a - FEMALES
12000 ppm: statistically significantly increased brain weight relative to body weight; statistically significantly decreased absolute spleen and thymus weight

F2b - MALES
12000 ppm: statistically significantly decreased absolute spleen weight but statistically significantly increased spleen weight relative to body weight; statistically significantly decreased absolute thymus weight

F2b - FEMALES
3000 ppm: statistically significantly increased brain weight relative to body weight; statistically significantly decreased absolute spleen and thymus weight
12000 ppm: statistically significantly decreased absolute brain weight but increased brain weight relative to body weight; statistically significantly decreased absolute spleen and thymus weight

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

12000 ppm: statistically significantly increased autolytic F2a pups

Histopathological findings:

not examined

Other effects:

no effects observed

Developmental neurotoxicity (F2)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F2)

Developmental immunotoxicity:

not examined

Details on results (F2)

MORTALITY / VIABILITY
Considering the fact that the highest viability was calculated each for F2a and F2b 12000 ppm pups and the lactation indices were not altered dose-dependently after both matings up to 12000 ppm the low viability was not considered treatment-related.

GROSS PATHOLOGICAL FINDINGS
Since no dose dependent increase in autolytic F2b pups was observed, the increase in autolytic F2a pups was not considered treatment-related.

Effect levels (F2)

1

Dose descriptor:

NOAEL

Remarks:

systemic toxicity

Generation:

F2a

Effect level:

3 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

organ weights and organ / body weight ratios

2

Dose descriptor:

NOAEL

Remarks:

systemic toxicity

Generation:

F2b

Effect level:

750 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

Target system / organ toxicity (F2)

Critical effects observed:

no

Overall reproductive toxicity

Reproductive effects observed:

no

Any other information on results incl. tables

Table 2: Mortality and clinical signs (numbers of animals affected) in parental animals

Observations	Dietary concentration (ppm)
	0	750	3000	12000	0	750	3000	12000
	P0 males				P1 males
Mortality No. of unscheduled deaths	0	0	0	0	0	1	0	0
	P0 females				P1 females
Mortality No. of unscheduled deaths	0	0	0	1	0	0	0	0
Clinical findings (lactation) Increased water intake	0	0	0	0	0	0	0	2

 Table 3: Mean body weight and food consumption – premating

Observations (study week)	Dietary concentration (ppm)
	0	750	3000	12000
P0 males (during premating)
Mean body weight (g) week 10 (about end of premating)	369.9 ± 21.42	369.5 ± 22.80	372.3 ± 19.87	332.5** ± 22.68
Mean weight gain (g) weeks (1-10)	241.0 ± 21.30	238.7 ± 19.82	239.3 ± 17.49	202.3** ± 20.82
Mean food consumption (g/kg body weight/day) weeks (1-10)	86.1 ± 29.48	85.0 ± 28.50	82.6 ± 29.01	86.9 ± 31.42
P0 females (during premating)
Mean body weight (g) week 10 (about end of premating)	219.8 ± 16.03	220.8 ± 12.37	217.9 ± 17.91	205.6** ± 13.99
Mean weight gain (g) weeks (1-10)	109.9 ± 11.97	113.2 ± 11.79	111.2 ± 18.48	98.7* ± 11.56
Mean food consumption (g/kg body weight/day) weeks (1-9)	101.0 ± 23.40	106.8 ± 25.29	99.4 ± 21.40	99.1 ± 25.26
P1 males (during premating)
Mean body weight (g) week of age 16 (end of premating)	406.2 ± 43.37	395.1 ± 48.91	394.1 ± 26.02	342.5** ± 20.76
Mean weight gain (g) weeks of age 5-16	308.7 ± 38.53	297.0 ± 42.01	300.0 ± 21.16	260.3** ± 21.64
Mean food consumption (g/kg body weight/day) week of age 5-15	99.6 ±54.74	99.4 ± 44.55	96.9 ± 45.79	100.4 ± 49.72
P1 females (during premating)
Mean body weight (g) week 16 of age (end of premating)	230.0 ±15.20	232.1 ±17.66	227.5 ± 10.44	204.3** ± 13.32
Mean weight gain (g) weeks of age 5-16	141.3 ± 12.27	143.3 ± 17.27	141.8 ± 12.72	129.2* ± 13.12
Mean food consumption (g/kg body weight/day) week of age 5-15	128.2 ± 59.16	120.6 ± 45.12	121.5 ± 42.42	105.3 ± 40.85

* statistically different from control, p ≤ 0.05, ** statistically different from control, p ≤ 0.01

 Table 4: Mean body weight and food consumption – gestation or lactation

Observations (study day)	Dietary concentration (ppm)
	0	750	3000	12000
P0 females (during gestation or lactation) → F1
Mean body weight on day 20 p.c. (g)	314.8 ± 23.87	315.5 ± 18.89	306.1 ± 26.06	283.1** ± 17.81
Mean weight gain day 14 to 20 p.c. (g)	51.4 ± 10.85	51.8 ± 8.46	49.4 ± 10.40	43.2* ± 6.03
Mean food consumption day 14 to 20 p.c. (g/kg body weight/day)	71.2 ± 4.58	72.2 ± 4.11	72.3 ± 4.87	71.8 ± 7.12
Mean body weight day 4 p.p. (g)	256.8 ± 19.02	256.3 ± 15.30	250.7 ± 18.76	226.1** ± 16.31
Mean weight gain day 0 to 4 p.p.  (g)	9.8 ± 10.77	4.4 ± 12.86	8.3 ± 11.66	4.3 ± 9.43
Mean food consumption day 0 to 4 p.p. (g/kg body.weight/day)	112.1 ± 27.99	118.9 ± 43.96	106.9 ± 36.84	115.5 ± 35.95
P1 females (during gestation or lactation) → F2a
Mean body weight on day 20 p.c. (g)	312.9 ± 30.32	321.3 ± 22.59	312.1 ± 25.36	283.1** ± 17.76
Mean weight gain day 14 to 20 p.c. (g)	43.1 ± 19.93	51.2 ± 7.77	47.8 ± 13.66	44.6 ± 6.91
Mean food consumption day 14 to 20 p.c. (g/kg body weight/day)	77.6 ± 22.19	74.4 ± 10.06	78.4 ± 11.85	81.9 ± 16.65
Mean body weight day 4 p.p. (g)	262.0 ± 13.94	257.7 ± 17.29	255.1 ± 13.11	223.0** ± 14.96
Mean weight gain day 0 to 4 p.p. (g)	3.7 ± 11.49	5.0 ± 11.82	0.8 ± 11.46	-4.9* ± 8.09
Mean food consumption day 0 to 4 p.p. (g/kg body weight/ day)	123.0 ± 47.72	110.7 ± 42.20	115.0 ± 41.82	131.9 ± 43.79
P1 females (during gestation or lactation) → F2b
Mean body weight on day 20 p.c. (g)	335.6 ±32.40	339.5 ± 37.17	343.5 ± 25.78	295.8** ± 26.14
Mean weight gain day 14 to 20 p.c. (g)	44.2 ± 26.94	49.3 ± 22.24	50.4 ± 20.79	41.1 ± 14.44
Mean food consumption day 14 to 20 p.c. (g/kg body weight/day)	62.8 ±13.76	62.1 ±9.04	65.0 ± 8.12	64.4 ± 8.35
Mean body weight day 4 p.p. (g)	290.3 ± 12.69	288.8 ± 22.67	279.7 ± 15.32	242.1** ± 15.44
Mean weight gain day 0 to 4 p.p. (g)	11.5 ± 8.43	7.5 ± 13.39	0.7** ± 11.71	2.8* ± 7.56
Mean food consumption day 0 to 4 p.p. (g/kg body weight/ day)	140.0 ± 56.11	115.6 ± 36.82	93.2** ± 28.00	124.6 ± 34.76

p.c. = postcoital, p.p. = postpartum

* statistically different from control, p ≤ 0.05, ** statistically different from control, p ≤ 0.01

 Table 5: Mean test substance intake during premating (mg/kg bw/day)

Observation in study weeks	Males			Females
	Dietary concentration (ppm)
	750	3000	12000		750	3000	12000
P0 (males: study week 1-10) (females: study week 1-9)	63.8 ± 21.38	247.8 ± 87.02	1043.0 ± 377.06		80.1 ± 18.96	298.2 ± 64.21	1189.7 ± 303.11
P1 (week of age 5-15)	74.5 ± 33.42	288.4 ± 137.38	1204.9 ± 596.63		90.4 ± 33.84	364.5 ± 127.27	1263.4 ± 490.23

 Table 6: Mean test substance intake during gestation and lactation (mg/kg bw/day)

Observation in study days	Dietary concentration (ppm)
	750	3000	12000
P0 females
Mean test substance consumption day 14to20 p.c.	54.2 ± 3.08	216.8 ± 14.60	861.8 ± 85.46
Mean test substance consumption day 0 to 4 p.p.	89.2 ± 32.97	320.7 ± 110.53	1385.8 ± 431.38
P1 females → F2a
Mean test substance consumption day 14 to 20 p.c.	55.8 ± 7.55	235.3 ± 35.55	982.3 ± 199.80
Mean test substance consumption day 0 to 4 p.p.	83.0 ± 31.65	345.1 ± 125.47	1582.8 ± 525.45
P1 females → F2b
Mean test substance consumption day 14 to 20 p.c.	46.6 ± 6.78	195.1        : ± 24.36	773.4 ± 100.17
Mean test substance consumption day 0 to 4 p.p.	86.7 ± 27.61	279.7 ± 84.01	1495.3 ± 417.14

p.c. = postcoital, p.p. = postpartum

 Table 7: Mean absolute organ weights (mg) of P0 animals

Observations	Dietary concentration (ppm)
	0	750	300	12000
P0 males
Body weight (g)	446.9 ± 25.87	445.8 ± 28.33	447.8 ± 29.46	409.5** ± 25.75
Kidneys	2982 ± 250.9	2917 ± 289.8	2926 ± 149.0	2818 ± 195.7
Liver	16208 ±1662.3	16040 ±1742.1	15928 ± 1212.0	14419** ± 1362.1
Spleen	714 ± 75.6	715 ± 75.6	732 ± 76.0	683 ± 75.4
Seminal vesicles	1805 ± 328.8	1758 ± 208.2	1654 ± 250.7	1548** ± 252.8
Testes	3437  ± 560.7	3548 ±251.3	3661 ± 268.6	3458 ± 263.5
Brain	1933 ± 65.5	1971 ± 64.6	1976 ± 74.1	1914 ± 60.7
Adrenals	64 ± 9.8	51** ± 10.3	50** ± 12.1	56** ± 5.7
Pituitary	13 ± 3.3	13  3.4	16** ± 3.9	12 ± 3.2
Thyroid	17 ± 5.0	17 ± 4.0	18 ± 3.5	18 ± 6.0
P0 females
Body weight (g)	255.4 ± 18.06	259.4 ± 12.21	253.8 ± 15.36	228.5** ± 13.64
Kidneys	2037 ± 156.8	2069 ± 125.0	2050 ± 123.1	1905** ± 148.4
Liver	12384 ± 1570.3	13227 ± 1291.1	12592 ± 1610.7	13160 ± 1097.4
Spleen	550 ± 57.9	537 ± 38.5	521 ± 68.0	451** ± 64.5
Ovaries	130 ± 33.7	135 ± 26.7	121 ± 26.0	91** ± 27.5
Uterus	522 ± 188.4	515 ± 104.4	525 ± 259.3	378* ± 139.0
Brain	1789 ± 59.4	1792 ± 73.5	1759 ± 67.3	1769 ±74.0
Adrenals	64 ± 12.0	66 ± 9.0	61 ± 10.6	58 ± 9.8
Pituitary	13 ± 3.0	14 ± 2.4	15* ± 3.3	12 ± 2.7
Thyroid	13 ± 3.9	15 ± 3.3	16 ± 3.9	16 ± 3.0

* statistically different from control, p ≤ 0.05, ** statistically different from control, p ≤ 0.01

 Table 8: Mean relative organ weights (mg/100 g bw) of P0 animals

Observations	Dietary concentration (ppm)
	0	750	300	1200
P0 males
Body weight (g)	446.9 ± 25.87	445.8 ±28.33	447.8 ±29.46	409.5** ± 25.75
Kidneys	667.0 ± 36.64	653.8 ± 41.13	655.2 ± 43.60	688.5 ± 29.93
Liver	3622.0 ± 232.95	3592.4 ± 244.99	3566.0 ± 300.07	3519.2 ± 218.28
Spleen	159.7 ± 13.93	160.7 ± 17.01	163.3 ± 12.24	166.8 ±15.73
Seminal vesicles	405.2 ± 74.56	395.7 ± 51.83	371.4 ± 65.16	378.7 ± 59.46
Testes	769.0 ± 121.14	798.2 ± 66.84	821.4 ± 85.15	846.2** ± 65.79
Brain	433.6 ± 23.53	443.4 ± 25.17	443.0 ± 31.02	468.8** ± 26.73
Adrenals	14.4 ± 2.21	11.4** ± 2.06	11.2** ± 2.75	13.6 ± 1.52
Pituitary	2.8 ± 0.77	2.9 ± 0.68	3.5** ± 0.97	3.0 ± 0.78
Thyroid	3.8 ± 1.09	3.9 ± 0.81	3.9 ± 0.70	4.5 ± 1.52
P0 females
Body weight (g)	255.4 ± 18.06	259.4 ± 12.21	253.8 ± 15.36	228.5** ± 13.64
Kidneys	799.3 ± 54.94	798.3 ± 49.52	808.6 ± 36.08	834.5* ± 50.08
Liver	4841.9 ± 449.21	5109.3 ± 558.83	4965.4 ± 581.05	5758.1** ± 295.61
Spleen	215.8 ± 21.76	207.4 ± 18.33	206.0 ± 29.77	197.5 ± 27.02
Ovaries	51.2 ± 13.45	51.8 ± 9.58	47.7 ± 9.21	39.9** ± 11.34
Uterus	205.2 ± 76.94	199.5 ± 45.76	206.0 ± 98.42	165.6 ± 59.64
Brain	703.5 ± 50.53	691.8 ± 35.42	695.4 ± 45.95	776.1** ± 40.16
Adrenals	25.3 ± 4.97	25.4 ± 3.48	24.0 ± 3.66	25.4 ± 4.04
Pituitary	4.9 ± 1.07	5.3 ± 0.94	5.8  ± 1.26	5.3 ± 1.16
Thyroid	5.2 ± 1.54	5.6  ± 1.32	6.3* ± 1.57	6.9** ± 1.36

* statistically different from control, p ≤ 0.05, ** statistically different from control, p ≤ 0.01

 Table 9: Mean absolute organ weights (mg) of P1 animals

Observations	Dietary concentration (ppm)
	0	750	300	12000
P1 males
Body weight (g)	494.0 ± 49.91	492.7 ± 27.73	486.4 ± 29.95	436.5** ± 25.66
Kidneys	3252 ± 389.2	3159 ± 216.4	3113 ± 265.6	2953** ± 223.9
Liver	18900 ± 3124.1	17809 ± 1580.6	17334* ± 1510.8	15264** ± 1717.9
Spleen	807 ± 109.4	782 ± 69.6	802 ± 69.7	718** ± 79.2
Seminal vesicles	1466 ± 362.6	1602 ± 259.3	1556 ± 180.7	1731** ± 312.3
Testes	3508  ± 886.2	3656 ± 234.6	3734 ± 217.3	3430 ± 390.4
Brain	1965 ± 77.4	2048** ± 64.1	1997 ± 67.6	1923 ± 72.8
Adrenals	55 ± 11.5	50 ± 7.3	50 ± 8.0	49 ± 7.6
Pituitary	11 ± 2.4	14** ± 5.3	13 ± 3.7	11 ± 2.9
Thyroid	15 ± 5.1	16 ± 4.2	15 ± 4.2	14 ± 2.6
P1 females
Body weight (g)	286.2 ± 15.98	286.9 ± 23.98	281.5 ± 16.68	246.7** ± 14.41
Kidneys	2151 ± 154.3	2204 ± 215.4	2170 ± 208.9	2128 ± 169.1
Liver	11641 ± 2275.5	13054 ± 2562.9	12857 ± 2485.2	13471 ± 2393.0
Spleen	555 ± 61.4	577 ± 83.5	587 ± 87.3	530 ± 65.2
Ovaries	142 ± 16.8	146 ± 21.2	143 ± 24.1	112** ± 25.3
Uterus	677 ± 275.1	631 ± 148.5	639 ± 180.9	480** ± 166.7
Brain	1854 ± 67.0	1849 ± 74.3	1853 ± 64.1	1794** ± 70.2
Adrenals	61 ± 7.3	62 ± 7.2	58 ± 6.5	58 ± 7.5
Pituitary	14 ± 3.8	16 ± 4.8	14 ± 4.1	12 ± 4.1
Thyroid	13 ± 3.5	12 ± 1.8	13 ± 3.4	12 ± 3.5

* statistically different from control, p ≤ 0.05, ** statistically different from control, p ≤ 0.01

 Table 10: Mean relative organ weights (mg/100 g bw) of P1 animals

Observations	Dietary concentration (ppm)
	0	750	3000	12000
P1 males
Body weight (g)	494.0 ± 49.91	492.7 ± 27.73	486.4 ± 29.95	436.5** ± 25.66
Kidneys	659.9 ± 59.24	642.0 ± 43.79	640.0 ± 39.32	676.6 ± 37.80
Liver	3825.6 ± 494.82	3619.0 ± 308.29	3566.9* ± 287.88	3495.0** ± 319.80
Spleen	163.4 ± 14.64	158.9 ± 13.67	165.1 ± 14.89	164.5 ± 14.10
Seminal vesicles	299.3 ± 77.00	325.0 ± 48.09	320.6 ± 38.55	396.8** ± 69.40
Testes	701.9 ± 167.47	743.1 ± 482.3	769.8 ± 56.71	786.9* ± 90.62
Brain	401.3 ± 38.66	416.7 ± 22.65	411.9 ± 25.83	441.6** ± 25.17
Adrenals	11.2 ± 3.09	10.1 ± 1.49	10.2 ± 1.66	11.2 ± 1.76
Pituitary	2.2 ± 0.51	2.9** ± 1.08	2.6 ± 0.81	2.5 ± 0.70
Thyroid	3.1 ± 1.06	3.2 ± 0.91	3.0 ± 0.78	3.2 ± 0.62
P1 females
Body weight (g)	286.2 ± 15.98	286.9 ± 23.98	281.5 ± 16.68	246.7** ± 14.14
Kidneys	753.2 ± 60.89	772.2 ± 85.09	772.8 ± 81.69	863.2** ± 59.84
Liver	4081.8 ± 830.57	4572.1 ± 917.36	4586.5 ± 940.91	5476.9** ± 1002.6
Spleen	194.1 ± 21.45	201.6 ± 25.76	208.9 ± 32.05	215.2* ± 26.30
Ovaries	49.7 ± 7.15	50.9 ± 6.02	50.9 ± 9.37	45.3 ± 9.69
Uterus	236.4 ± 93.19	221.8 ± 58.69	227.2 ± 62.82	194.2 ± 64.33
Brain	649.3 ± 34.63	647.5 ± 44.15	660.2 ± 43.06	728.9** ± 41.26
Adrenals	21.5 ± 2.81	21.6 ± 2.97	20.6 ± 2.13	23.6* ± 3.33
Pituitary	4.8 ± 1.41	5.7 ± 1.80	5.0 ± 1.44	4.7 ± 1.76
Thyroid	4.4 ± 1.19	4.3 ±0.65	4.7 ±1.18	4.9  ±1.39

* statistically different from control, p ≤ 0.05, ** statistically different from control, p ≤ 0.01

 Table 11: Remarkable necropsy findings of P0 and P1 females

Observation	No. of animals affected
	Dietary concentration (ppm)
	0	750	3000	12000	0	750	3000	12000
	P0 females				P1 females
Ovaries - Diminished in size	-	-	-	2	-	-	-	-
Stomach - Dilatation	-	-	-	-	-	-	-	3
Cecum - Dilatation	-	-	-	-	-	-	-	4
Emaciation	-	-	-	-	-	-	-	7

Table 12: Remarkable histopathological findings of P0 and P1 females

Observation	No. of animals affected
	Dietary concentration (ppm)
	0	750	3000	12000	0	750	3000	12000
	P0 males				P1 males
Liver
- Cytoplasmic change (pp)	5	9	5	20	-	1	1	10
- Reduced glycogen content	2	2	-	11	2	3	1	5
- Fat deposition (pp)	20	22	23	7	22	22	22	13
- Fat deposition (diffuse)	5	3	2	18	1	3	3	12
Kidneys
- Necrosis (pap)	1	-	-	-	-	-	-	1
- Simple tubulus dilation (pap)	3	-	1	-	5	2	3	10
- Basophilic tubules	20	20	20	21	24	23	25	22#
- Hyaline casts/tubulus dilation	14	9	9	7	22	20	23	4
	P0 females				P1 females
Ovaries
- Atrophy	-	-	-	5	-	-	-9
Vagina
- Metestrus	7	4	5	15	4	5	5	10
- Diestrus	9	10	9	2	4	11	10	1
- Atrophie epithelium	1	2	1	16	8	3	3	14
Liver
- Cytoplasmic change (pp)	-	-	-	-	2	-	-	-
- Hypertrophy /eosinophilia (pp)	-	-	-	10	0	2	10	13
- Reduced glycogen content	6	3	7	8*	2	2	1	9
- Fat deposition (pp)	1	2	1	2	20	23	17#	15#
- Fat deposition (diffuse)	2	3	3	-	5	-	5	4
Kidneys
- Necrosis (pap)	-	-	-	-	-	-	-	5
- Simple tubulus dilation (pap)	1	2	-	3	-	-	2	12
- Increased tubulus epithel inclusion	10	10	12	24	10	12	14	23
- Tubulus dilation (cm)	0	0	0	22	0	0	2	23
- Basophilic tubules	7	6	5	16	14	17	20*	19*
- Hyaline casts/tubulus dilation	-	-	3	-	2	1	5	1

pap = papilla, pp = periportal, cm = corticomedullary

* increased by grading, # reduced by grading, - no finding

Table 13: Litter parameters for P0 generation

Observation	Dietary concentration (ppm)
	0	750	3000	12000
	→ F1 pups
Mean implantations^a	11.96    ± 2.368	11.28    ± 1.696	11.68    ± 2.495	11.58    ± 1.558
No. of females with implantations^a	24	25	25	25
Mean prenatal loss^a	0.92     ± 1.176	0.88    ± 0.971	0.80 ± 1.080	1.71   ± 1.488
Number born	265	260	272	237
Number born dead	0	2	0	1
Live birth index	100.00	99.43	100.00	99.54
%males on Day 0	50.92	57.98	49.69	51.49
Viability index	99.58	99.24	92.97	98.05
Deathson Days 0-4(%)	0.4	0.8	7.7	1.3
Lactation index	96.88	100.00	98.44	100.00
Deaths on Days 5-21(%)	2.3	0.0	1.1	0.0
Mean litter size :
Day 0	11.04   ± 2.458	10.32   ± 1.909	10.88    ± 2.386	9.83     ± 1.659
Day 4^b	11.00    ± 2.485	10.24    ± 1.921	10.46   ± 2.621	9.67     ± 1.810
Day 4^c	7.83    ± 0.816	7.76    ± 0.831	7.63    ± 1.279	7.75    ± 0.608
Day 7	7.79 ± 0.833	7.76    ± 0.831	7.58    ± 1.283	7.75 ± 0.608
Day 14	7.58 ± 1.176	7.76    ± 0.831	7.54 + 1.285	7.75    ± 0.608
Day 21	7.58    ± 1.176	7.76    ± 0.831	7.50    ± 1.285	7.75    ± 0.608

^aper litter,^bbefore culling,^cafter culling

 Table 14: Litter parameters for P1 generation

Observation	Dietary concentration (ppm)
	0	750	3000	12000
→ F2a pups
Mean implantations	Not determined after this mating
Mean prenatal loss	Not to calculate as two matings were performed
Number born	214	264	254	266
Number born dead	1	5	0	1
Live birth index	99.60	98.34	100.00	99.60
% males on Day 0	47.75	51.54	50.47	47.12
Viability index	74.86	93.53	79.73	85.67
Deathson Days 0-4(%)	21.1	6.5**	21.3	14.7
Lactation index	86.05	93.48	86.25	94.24
Deathson Days 5-21(%)	6.1	4.6	7.9	3.8
Mean litter size:
Day 0	10.14    ± 3.183	11.30    ± 1.743	11.04    ± 1.870	10.60    ± 1.848
Day 4^b	8.84    ± 3.862	10.57    ± 1.927	10.00    ± 2.340	10.27    ± 2.051
Day 4^c	6.79   ± 2.529	7.87**    ± 0.626	7.70   ± 0.733	7.82*  ± 0.395
Day 7	6.72    ± 2.296	7.48    ± 1.275	7.05    ± 1.761	7.59    ± 0.734
Day 14	6.50    ± 2.431	7.35    ± 1.335	6.70    ± 1.922	7.41    ± 0.959
Day 21	6.44 ±2.431	7.35    ± 1.335	6.70   ± 1.922	7.36 ± 1.002
→ F2b pups
No. of females with implantations^a	22	24	25	25
Mean prenatal loss^a	Not to calculate as two matings were performed
Number born	201	251	254	229
Number born dead	0	1	3	0
Live birth index	100.00	99.55	98.76	100.00
% males on Day 0	47.67	53.76	48.91	47.94
Viability index	81.95	96.09	82.46	94.86
Deathson Days 0-4(%)	20.4	4.0**	19.5	5.7**
Lactation index	74.09	78.98	74.55	76.51
Deathson Days 5-21(%)	16.4	14.8	15.9	18.8
Mean litter size:
Day 0	10.58    ± 2.714	11.36    ± 1.706	10.46    ± 2.519	9.54    ± 2.502
Day 4^b	9.41    ± 2.980	10.91    ± 2.348	9.62    ± 2.674	9.00    ± 2.485
Day 4^c	7.41    ± 1.228	7.82    ± 0.853	7.52    ± 0.873	7.42    ± 1.316
Day 7	6.00    ± 2.475	6.64    ± 2.237	6.30    ± 2.342	6.57    ± 2.019
Day 14	5.81    ± 2.228	6.18    ± 2.42	95    ± 2.373	5.87    ± 2.201

^aper litter,^bbefore culling,^cafter culling

* statistically different from control, p ≤ 0.05, ** statistically different from control, p ≤ 0.01

Table 15: Mean F1, F2a and F2b pup weights (g)

Lactation day	Dietary concentration (ppm)
	0	750	3000	12000	0	750	3000	12000
	F1 pups - male				F2a pups - male
0	5.65 ± 0.503	5.74 ± 0.510	5.50 ± 0.428	5.72 ± 0.510	5.73 ± 0.401	5.65 ± 0.421	5.66 ± 0.637	5.57 ± 0.616
4^b	9.13 ± 1.418	9.32 ± 1.066	9.05 ± 1.053	9.37 ± 0.975	8.65 ± 1.948	8.72 ± 1.508	8.29 ± 2.010	8.50 ± 1.141
4^c	9.13 ± 1.427	9.33 ± 1.082	9.09 ± 1.048	9.39 ± 0.974	8.67 ± 1.956	8.73 ± 1.482	8.31 ± 2.063	8.49 ± 1.161
7	14.19 ± 2.325	14.21 ± 1.400	14.29 ± 1.514	13.50 ± 1.144	13.30 ± 2.745	14.10 ± 1.817	12.79 ± 2.932	12.22 ± 1.901
14	27.97 ± 3.078	28.44 ± 2.292	28.59 ± 2.168	24.18** ± 1.894	27.86 ± 5.562	29.04 ± 2.357	27.75 ± 3.133	24.07** ± 2.353
21	43.86 ± 3.869	44.19 ± 3.239	43.41 ± 3.230	35.57** ± 2.666	44.37 ± 6.284	45.43 ± 3.351	43.62 ± 4.534	35.76** ± 2.966
28	75.82 ± 5.706	76.53 ± 5.945	75.28 ± 4.907	61.79** ± 3.811	76.97 ± 8.864	78.38 ± 4.305	74.90 ± 7.139	61.96** ± 4.393
	F1 pups - female				F2a pups - female
0	5.31 ± 0.440	5.40 ± 0.509	5.28 ± 0.433	5.31 ± 0.504	5.35 ± 0.477	5.34 ± 0.431	5.35 ± 0.604	5.21 ± 0.542
4^b	8.62 ± 1.204	8.86 ± 1.116	8.49 ± 1.242	8.91 ± 8.94	8.42 ± 1.758	8.22 ± 1.528	7.99 ± 1.780	8.00 ± 1.095
4^c	8.65 ± 1.256	8.91 ± 1.087	8.49 ± 1.245	8.96 ± 0.938	8.45 ± 1.844	8.27 ± 1.543	8.04 ± 1.774	8.07 ± 1.130
7	13.56 ± 1.948	13.63 ± 1.560	13.28 ± 1.690	12.82 ± 1.003	12.56 ± 2.227	13.17 ± 2.277	12.64 ± 2.579	11.82 ± 1.890
14	27.31 ± 3.030	27.63 ± 2.630	26.88 ± 2.385	23.31** ± 1.845	27.44 ± 4.292	28.46 ± 2.893	27.02 ± 3.620	23.93** ± 2.202
21	42.60 ± 4.275	43.32 ± 3.808	41.28. ± 3.452	34.24** ± 2.440	42.35 ± 5.857	44.18 ± 4.075	42.21 ± 4.494	35.12** ± 2.505
28	70.33 ± 5.386	71.21 ± 4.652	68.82 ± 4.606	58.42** ± 3.395	69.54 ± 7.557	72.05 ± 5.164	68.84 ± 6.822	58.54** ± 3.688
	F1 pups - female				F2a pups - female
0	5.96 ± 0.649	5.79 ± 0.615	5.49* ± 0.526	5.86 ± 0.483	5.63 ± 0.562	5.48 ± 0.646	5.19 ± 0.579	5.50 ± 0.422
4^b	8.59 ± 1.972	8.24 ± 1.693	7.79 ± 1.834	8.62 ± 1.759	8.35 ± 1.828	7.99 ± 1.611	7.54 ± 1.674	8.43 ± 1.903
4^c	8.63 ± 2.004	8.33 ± 1.723	7.85 ± 1.850	8.67 ± 1.727	8.36 ± 1.829	8.05 ± 1.603	7.55 ± 1.667	8.42 ± 1.900
7	12.60 ± 3.988	13.21 ± 2.880	12.80 ± 2.136	12.25 ± 2.295	13.89 ± 1.993	12.41 ± 2.706	11.74 ± 2.313	12.45 ± 2.961
14	28.48 ± 5.300	28.69 ± 4.385	27.99 ± 4.447	26.33 ± 3.745	31.07 ± 6.101	27.45 ± 5.813	25.72* ± 6.506	25.98* ± 4.861
21	46.09 ± 6.317	46.20 ± 5.817	42.96 ± 5.832	39.19** ± 4.944	48.83 ± 8.419	45.37 ± 5.031	41.33** ± 7.091	39.03** ± 7.219
28	80.03 ± 10.369	80.81 ± 7.766	75.73 ± 7.905	68.14** ± 6.705	78.49 ± 6.827	75.82 ± 6.603	68.97** ± 7.858	64.28** ± 8.539

^bbefore culling,^cafter culling

* statistically different from control, p ≤ 0.05, ** statistically different from control, p ≤ 0.01

 Table 16: Sexual maturation of F1 post weanlings

Observation	Dietary concentration (ppm)
	0	750	3000	12000
F1 generation
Balano-preputial separation
at mean age (days)	42.4 ± 3.04	43.1 ± 5.95	43.0 ± 3.21	44.0 ± 3.58
at mean body weight (g)	162.4 ± 18.57	159.5 ± 18.12	159.2 ± 16.32	141.1** ± 16.33
Vaginal opening
at mean age (days)	33.9 ± 1.89	34.4 ± 1.98	36.4** ± 1.78	36.8** ± 2.84
at mean body weight (g)	95.0 ± 10.93	95.8 ± 9.37	99.5 ± 7.53	91.3 ± 9.75

* statistically different from control, p ≤ 0.05, ** statistically different from control, p ≤ 0.01

 Table 17: Mean absolute organ weights (mg) of F1, F2a and F2b weanlings

Observations	Dietary concentration (ppm)
	0	750	300	12000
F1 pups – males
Body weight (g)	77.0 ± 8.51	78.6 ± 8.05	77.1 ± 6.87	63.9** ± 5.70
Brain	1444 ± 71.0	1473 ± 65.7	1437 ± 46.8	1399* ± 67.3
Spleen	251 ± 39.3	253 ± 41.7	249 ± 28.9	187** ± 24.4
Thymus	321 ± 64.1	347 ± 59.9	314 ± 51.0	281 ± 57.5
F1 pups – females
Body weight (g)	72.5 ± 7.04	73.2 ± 5.90	69.6 ± 6.65	59.1** ± 5.70
Brain	1380 ± 118.5	1415 ± 79.8	1373 ± 62.9	1312** ± 82.6
Spleen	234 ± 30.8	229 ± 29.3	220 ± 34.4	172** ± 19.2
Thymus	327 ± 83.0	332 ± 55.3	306 ± 57.4	164** ± 50.1
Uterus	72 ± 29.6	76 ± 26.4	73 ± 26.7	65 ± 24.1
F2a pups – males
Body weight (g)	78.2 ± 10.42	80.4 ± 7.16	75.5 ± 11.04	62.0** ± 8.82
Brain	1416 ± 83.5	1416 ± 96.2	1432 ± 75.4	1345* ± 62.9
Spleen	256 ± 28.3	278 ± 24.6	255 ± 42.7	195** ± 33.0
Thymus	365 ± 58.9	345 ± 71.1	331 ± 52.5	265** ± 45.5
F2a pups – females
Body weight (g)	72.5 ± 9.10	74.3 ± 6.98	72.2 ± 6.75	60.2** ± 6.11
Brain	1374 ± 68.4	1368 ± 76.4	1337 ± 82.4	1333 ± 77.8
Spleen	233 ± 36.0	239 ± 28.7	241 ± 38.4	186** ± 30.5
Thymus	356 ± 62.7	343 ± 70.7	346 ± 48.5	261** ± 55.6
Uterus	56 ± 19.9	62 ± 22.6	55 ± 18.6	52 ± 18.0
F2b pups – males
Body weight (g)	80.6 ± 12.41	82.2 ± 11.09	78.8 ± 9.67	74.9 ± 10.04
Brain	1408 ± 90.6	1416 ± 82.9	1432 ± 68.4	1404 ± 40.3
Spleen	303 ± 21.7	300 ± 44.5	285 ± 54.3	245** ± 38.0
Thymus	397 ± 57.3	375 ± 71.8	368 ± 69.3	320** ± 57.0
F2b pups – females
Body weight (g)	80.5 ± 9.18	77.5 ± 9.17	70.3** ± 8.45	68.0** ± 9.32
Brain	1403 ± 54.4	1388 ± 62.7	1359 ± 52.3	1350* ± 57.9
Spleen	267 ± 24.9	266 ± 38.2	238* ± 28.8	216** ± 41.3
Thymus	406 ± 59.9	364 ± 60.3	356* ± 46.8	300** ± 57.5
Uterus	65 ± 17.7	64 ± 14.6	67 ± 13.5	61 ± 18.7

* statistically different from control, p ≤ 0.05, ** statistically different from control, p ≤ 0.01

 Table 1: Mean relative organ weights (mg/100 g bw) of F1, F2a and F2b weanlings

Observation	Dietary concentration (ppm)
	0	750	3000	12000
F1 pups – males
Body weight (g)	77.0 ± 8.15	78.6 ± 8.05	77.1 ± 6.87	63.9**  ± 5.70
Brain	1888.2  ± 142.02	1887.6 ± 150.87	1874.6 ± 149.6	2204.2*  ± 195.41
Spleen	327.0 ± 42.34	321.5 ± 38.86	324.0 ± 42.20	291.7**  ± 23.60
Thymus	417.3 ± 69.80	441.2 ± 67.14	408.4 ± 61.32	438.7  ± 82.96
F1 pups – females
Body weight (g)	72.5  ± 7.04	73.2 ± 5.90	69.6 ± 6.65	59.1**  ± 3.79
Brain	1911.5  ± 170.29	1942.5 ± 166.88	1988.0 ± 187.16	2224.5*  ± 156.02
Spleen	322.4  ± 29.34	313.2 ± 36.07	316.6 ± 442.3	291.2**  ± 28.91
Thymus	446.7  ± 86.35	454.8 ± 75.93	440.2 ± 75.90	446.7  ± 87.56
Uterus	99.1 ± 38.16	103.5 ± 34.35	106.0 ± 40.49	110.3  ± 39.33
F2a pups – males
Body weight (g)	78.2 ± 10.42	80.4 ± 7.16	75.5 ± 11.04	62.0**  ± 8.82
Brain	1836.5  ± 232.91	1770.8 ± 156.48	1926.5 ± 227.58	2207.3*  ± 283.27
Spleen	331.0 ± 41.06	346.3 ± 26.04	341.0 ± 58.87	316.0  ± 42.04
Thymus	467.5  ± 49.12	429.4 ± 84.26	442.7 ± 69.24	433.0  ± 83.04
F2a pups – females
Body weight (g)	72.5  ± 9.10	74.3 ± 6.98	72.2 ± 6.75	60.2**  ± 6.11
Brain	1917.5 ± 218.42	1849.9 ± 139.52	1860.6 ± 125.13	2229.7*  ± 193.46
Spleen	324.1  ± 51.53	321.9 ± 30.51	335.7 ± 54.07	310.0  ± 44.50
Thymus	490.7  ± 64.19	461.3 ± 88.31	480.3 ± 62.99	432.5  ± 75.29
Uterus	76.1  ± 23.65	84.4 ± 33.56	76.3 ± 25.62	87.0  ± 29.31
F2b pups – males
Body weight (g)	80.6  ± 12.41	82.2  ± 11.09	78.8 ± 9.67	74.9  ± 10.04
Brain	1773.88 ± 208.21	1743.9 ± 169.88	1842.0 ± 220.87	1900.4  ± 222.11
Spleen	383.6  ± 62.69	367.8 ± 45.76	363.0 ± 54.77	327.2**  ± 38.45
Thymus	498.0  ± 78.38	460.6 ± 93.50	467.8 ± 79.80	427.7  ± 58.76
F2b pups – females
Body weight (g)	80.5  ± 9.18	77.5 ± 9.17	70.3** ± 8.45	68.0** ± 9.32
Brain	1757.7 ± 152.10	1808.8 ± 180.72	1953.4* ± 191.11	2017.2** ± 240.58
Spleen	333.9  ± 31.81	345.3 ± 46.12	341.1 ± 373.7	320.9  ± 64.42
Thymus	507.4  ± 76.13	471.2 ± 70.94	508.5 ± 56.94	442.7*  ± 67.08
Uterus	80.4  ± 20.85	82.8 ± 19.89	96.2 ± 20.83	89.7  ± 25.21

* statistically different from control, p ≤ 0.05, ** statistically different from control, p ≤ 0.01

Applicant's summary and conclusion

Executive summary:

The influence of the test substance on reproductive performance was assessed in a two generation reproductive toxicity study in Wistar Crl: (WI) WU BR rats performed according to OECD Guideline 416 and in compliance with GLP. For the P0 generation, three groups of 25 male rats received the test substance orally, via the diet, at concentrations of 750, 3000 or 12000 ppm for ten weeks before pairing until termination. Four groups of 25 female rats received the test substance orally, via the diet, at concentrations of 750, 3000 or 12000 ppm for ten weeks before pairing, throughout pairing and gestation and during lactation. A similarly constituted control group received untreated basal diet for the same duration. The F1 offspring were nursed up to an age of four weeks. 25 male and 25 female progeny from each group were designated for the P1 generation for breeding the F2a generation and they continued to receive the relevant diet, as per the P0 generation, throughout the study until termination. A second mating was done on P1 rats to breed the F2b to clarify relatively low viability indices at 0 and 3000 ppm observed in the F2a generation. Both F2 generations were nursed up to an age of four weeks.

During the 10 week pre-mating period the test compound intake at 750, 3000 and 12000 ppm was 638, 247.8 or 1043.0 mg/kg bw/day in males and 80.1, 298.2 or 1189.7 mg/kg bw/day in females of the P0 generation, respectively. The corresponding test compound intake for the P1 generation was 74.5, 288.4 or 1204.9 mg/kg bw/day in males and 904, 364.5 or 1263.4 mg/kg bw/day in females, respectively. During gestation and lactation test compound intake in females of the P0 generation was 54.2, 216.8 or 861.8 and 89.2, 320.7 or 1385.8 mg/kg bw/day, respectively. In P1 females generating and weaning the F2a generation test compound intake was 55.8, 235.3 and 982.3 mg/kg bw/day during gestation as well as 83.0, 345.1 and 1582.8 mg/kg bw/day during lactation. Test compound intake in P1 females generating and weaning the F2b generation was 46.6, 195.1 or 773.4 mg/kg bw/day during gestation and 86.7, 279.7 and 1495.3 mg/kg bw/day during lactation.

Mortality, clinical signs, body weights and food intake as well as reproduction parameters such as mating performance, fertility, gestation, rearing, oestrus cycling and sperm analyses were examined in P0 and P1 rats. Furthermore, litter parameters such as litter size, percentage of males born and pup weight at birth as well as viability and lactation indices, body weight gain and clinical signs were studied in F1, F2a and F2b offspring. Developmental milestones were evaluated in F1 post weanlings and ano-genital distance was measured in F2a pups. Necropsies were done on all rats. Implantation sites in P0 and P1 females were recorded. Selected organs were weighed in adult rats and weanlings (recorded as absolute weight and relative to body weight) and histopathology including ovarian follicle staging (only P1) was performed in a number of organs of P0 and P1 rats.

No changes in clinical signs of parental rats were evident up to 12000 ppm.

In the P0 and P1 males and females administered 12000 ppm test substance statistically significantly decreased body weights and/or weight gains were noted during the pre-mating period as well as gestation and lactation period (females). At 3000 ppm statistically significantly decreased mean body weight gain in females weaning the F2b generation was observed during lactation. While no effects on food consumption was observed in the P0 generation, decreased food consumption was observed in P1 females during lactation at 3000 ppm (statistically significant) and pre-mating at 12000 ppm without statistical significance. Additionally, 2/25 females of the P1 generation showed increased water intake during lactation (qualitatively determination). At necropsy smaller ovaries were observed in 2/25 females of the P0 generation at 12000 ppm. Emaciation was noted in 7/25 P1 females. 3/25 and 4/25 P1 females showed dilated stomach and cecum, respectively.

Statistically significant, treatment-related effects on organ weights evident as increased relative liver weight and decreased absolute and relative ovaries weight were noted for P0 females of the highest dose group only. In P1 males statistically significant, treatment-related decreased absolute and relative liver weight was noted at 3000 and 12000 ppm. Absolute and relative seminal vesicle weights were increased at 12000 ppm. In P1 females statistically significantly and treatment-related increased relative kidney, liver and spleen as well as decreased absolute ovaries weight was observed at 12000 ppm.

Histopathology revealed periportal cytoplasmic change in the liver, often coincided with reduced hepatocellular glycogen storage in 12000 ppm males of both parental generations. Simultaneously, periportal fat storage was reduced in favour of a more diffuse pattern. In contrast, adaptive periportal hypertrophy/eosinophilia occurred in P0 females at 12000 ppm and in P1 females increasingly at 3000 ppm and above. Reduced hepatocellular glycogen storage was also observed in these animals. Changes in the fat storage became slightly evident in P1 females at 3000 ppm and above (not statistically significant). These liver changes associated with the observed effects on the liver weight might reflect secondary catabolic effects in course of a significant body weight loss in high dose animals. In the kidneys, papillary necrosis was found in males and females of the P1 generation at 12000 ppm. Simple dilation of the papillary tubules was increased at 12000 ppm in both male parental generations and in the female P1 generation at 3000 ppm and above. These findings are accompanied with increased relative kidney weight in females and are interpreted as adverse. Brownish inclusions in the proximal tubules and dilated cortico-medullary tubules were only found in females and raised at 12000 ppm in both generations. A small number of P1 females also showed dilated cortico-medullary tubules at 3000 ppm. Secondary changes belonging to chronic progressive nephropathy (CPN) either increased (P0 females: basophilic tubules) or decreased (P0/P1 males: hyaline casts/dilated tubules; P1 males: basophilic tubules) under high dose treatment. Further histopathological findings in high dose females of both parental generations included ovarian atrophy, increased metoestrus, decreased dioestrus and atrophy of the vaginal epithelium. Histopathological evaluations of ovarian follicles and corpora lutea revealed a statistically significant decrease in the number of growing follicles and corpora lutea in P1 rats treated with 12000 ppm. These findings are discussed as possible secondary due to weight loss and are correlated with reduced ovary weights and/or smaller ovaries.

The parameters of the reproductive performance such as insemination, fertility, gestation and rearing indices as well as gestation length were not influenced by the treatment with the test substance up to 12000 ppm. There was no test substance-related reduction in viability and lactation indices up to 12000 ppm. At 12000 ppm depressed pup and litter weights occurred in all generations. At 3000 ppm slightly reduced body weights were observed for female F2b pups. The occurrence of developmental milestones (balano-preputial separation and vaginal opening) was delayed in 12000 ppm F1 rats. No effect was seen at measurements of the ano-genital distance in F2a pups. At 12000 ppm F2a pups exhibited respiration sounds and blue discolorations and more autolytic F2a pups were found than in the other groups. The spleen and thymus weights were decreased in nearly all pup generations at 12000 ppm. Thus, under the conditions of this study, it is considered that 3000 ppm (equivalent to the mean achieved dose before pairing: 247.8 mg/kg bw/day for P0 males; 298.2 mg/kg bw/day for P0 females) represents the NOAEL in this study for the P0 animals and the F1 and F2a offspring. For the P1 adult animals and the F2b offspring the corresponding NOAEL is 750 ppm (equivalent to 74.5 and 90.4 mg/kg bw/day for P1 males and P1 females, respectively).In terms of reproductive effects, no treatment-related findings were observed at any dose level tested in the P0 and P1 generation; therefore, the high dose of 12000 ppm (equivalent to the mean achieved dose before pairing: 1043.0 mg/kg bw/day for P0 males; 1189.7 mg/kg bw/day for P0 females; 1204.9 mg/kg bw/day for P1 males; 1263.4 mg/kg bw/day for P1 females) is considered to be the NOAEL for reproductive toxicity with regard to the parental generations. For the F1 offspring The NOAEL for reproductive toxicity is considered to be 3000 ppm.