Benzamide, N-[[4-[(cyclopropylamino)carbonyl]phenyl]sulfonyl]-2-methoxy-

Toxicity to reproduction

Administrative data

Endpoint:

two-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

11 Oct 2004 - 13 Jul 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

Qualifier:

according to guideline

Guideline:

OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)

Version / remarks:

adopted 22 Jan 2001

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

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

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

485-320-2

EC Name:

-

Cas Number:

221667-31-8

Molecular formula:

C18H18N205S

IUPAC Name:

N-[4-(cyclopropylcarbamoyl)benzenesulfonyl]-2-methoxybenzamide

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
- Age at study initiation: (P) 6-7 wks; (F1) 4 wks
- Weight at study initiation: (P) Males: 108-159 g; Females: 74-100 g, (F1) Males: 73-121 g; Females 60-109 g
- Fasting period before study: Not applicable, feeding study
- Housing: Singly except when co-housed for matings or with litters in Makrolon cages Type IIIh on low-dust soft-wood shavings or nesting material (day 20 p.c. to 14 p.p.).
- Diet: Kliba 3883.9.25 (Provimi Kliba SA, CH-4303 Kaiseraugst, Switzerland), ad libitum.
- Water: Tap water, ad libitum.
- Acclimation period: About 7 days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23 ± 2
- Humidity (%): 55 ± 5
- Air changes (per hr): Minimum of 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): Dietary formulations were prepared by mixing appropriate amounts of test substance with diet (Kliba 3883.9.25) using a pre-mixture. A mixing granulator was used (manufactured by the company Loedige, Paderborn, Germany).
- Storage temperature of food: At room tempearture.

Details on mating procedure:

- M/F ratio per cage: 1/1
- Length of cohabitation: A maximum of 12 times during the three-week mating period; inseminated females were not further co-housed.
- Proof of pregnancy: Vaginal plug/sperm in vaginal smear referred to as Day 0 of pregnancy.
- After 14 days of unsuccessful pairing (lack of weight gain following insemination): Females 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. According to the test laboratories experience, occurrence of sperms after the first co-housing day without a following pregnancy can happen, if an inexperienced male, co-housed with a female for the first time, inseminated the female outside the oestrus.
- Further matings after two unsuccessful attempts: Yes, Six (P) females of the 12000 ppm group, which did not get pregnant with the males appointed, were co-housed additionally over the whole day with another male of the same group, which had been shown to be fertile. These unscheduled co-housings were done to investigate the cause of lack of pregnancy of these females. On these females vaginal smears were taken for evaluation for positive sperm findings as done normally to prove a mating. Sperm positive (P) females were kept then individually until littering. Litters were killed when born, but no pup necropsies of these animals were done. The (P) females in question were necropsied when it became clear that they were not pregnant or after their littering. No other data than date of positive sperm finding and littering were recorded. Implantation sites of these females, if any, were not entered to the statistical analyses.
- After successful mating each pregnant female was caged singly in Makrolon cages Type IIIh on nesting material.
- Any other deviations from standard protocol: Generally, if a male had to be necropsied prior to co-housing the next male was co-housed with two females.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Checks on homogeneity and stability (low and high concentrations) and contents of active ingredient (all doses including control) were done at several times during the study. The dietary levels of the test substance were verified by HPLC-UV.
Homogeneity analysis: In samples taken from the top, middle and bottom of 100 and 15000 ppm mixtures the test substance was proven to be homogeneously distributed.
Stability analysis: The test substance concentration in 100 and 15000 ppm mixtures was within the range of ± 20% of their initially measured concentration after storage at animal room temperature for up to 15 days and after 15 days of freezer storage (95-104%).
Concentration analysis: The absence of the test substance in the control diet was confirmed. The test substance concentration (all concentrations including control) measured at four time points was in the range of ± 20% of the nominal concentrations (104-113%). The active ingredient at the low and high concentration was homogenously distributed.

Duration of treatment / exposure:

(P) Males: About 12 weeks before mating, 3 weeks during mating.
(P) Females: About 12 weeks before mating, 3 weeks during mating, about 22 days during gestation, 28 days during lactation until weaning.
(F1) Males: 28 days until weaning, during growth into adulthood (at least 10 weeks), mating and production of an F2 generation, until weaning (28 days) of the F2 generation.
(F1) Females: 28 days until weaning, during growth into adulthood (at least 10 weeks), mating and production of an F2 generation, until weaning (28 days) of the F2 generation.

Frequency of treatment:

Daily, 7 days/week

Details on study schedule:

- F1 parental animals not mated until 10 weeks after selected from the F1 litters.
- Selection of parents from F1 generation when pups were 28 days of age.
- Age at mating of the mated animals in the study: 18-19 weeks (P); 14 weeks (F1)

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

480 ppm

Remarks:

corresponding to actual dose ingested during the pre-mating period:
39.2 mg/kg bw/day for P generation males (P0)
55.3 mg/kg bw/day for P generation females (P0)
50.3 mg/kg bw/day for F1 parental males (P1)
68.5 mg/kg bw/day for F1 parental females (P1)

corresponding to actual dose ingested during gestation and lactation:
37.2 mg/kg bw/day for P generation females (P0), Days 14 to 20 post coitum
58.5 mg/kg bw/day for P generation females (P0), Days 0 to 4 post partum
42.0 mg/kg bw/day for F1 parental females (P1), Days 14 to 20 post coitum
60.5 mg/kg bw/day for F1 parental females (P1), Days 0 to 4 post partum

Doses / concentrations 2

Dose / conc.:

2 400 ppm

Remarks:

corresponding to actual dose ingested during the pre-mating period:
202.3 mg/kg bw/day for P generation males (P0)
260.3 mg/kg bw/day for P generation females (P0)
251.5 mg/kg bw/day for F1 parental males (P1)
323.7 mg/kg bw/day for F1 parental females (P1)

corresponding to actual dose ingested during gestation and lactation:
173.1 mg/kg bw/day for P generation females (P0), Days 14 to 20 post coitum
246.4 mg/kg bw/day for P generation females (P0), Days 0 to 4 post partum
196.2 mg/kg bw/day for F1 parental females (P1), Days 14 to 20 post coitum
272.6 mg/kg bw/day for F1 parental females (P1), Days 0 to 4 post partum

Doses / concentrations 3

Dose / conc.:

12 000 ppm

Remarks:

corresponding to actual dose ingested during the pre-mating period:
1006.9 mg/kg bw/day for P generation males (P0)
1350.2 mg/kg bw/day for P generation females (P0)
1292.4 mg/kg bw/day for F1 parental males (P1)
1696.5 mg/kg bw/day for F1 parental females (P1)

corresponding to actual dose ingested during gestation and lactation:
840.5 mg/kg bw/day for P generation females (P0), Days 14 to 20 post coitum
1370.8 mg/kg bw/day for P generation females (P0), Days 0 to 4 post partum
1001.2 mg/kg bw/day for F1 parental females (P1), Days 14 to 20 post coitum
1449.1 mg/kg bw/day for F1 parental females (P1), Days 0 to 4 post partum

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 a one-generation pilot study (Bayer Report AT02084, May 20, 2005), where rats received 1000, 4000 and 12000 ppm in the diet. In this study the NOEL was 1000 ppm in males and 4000 ppm in females. At higher concentrations the body weight gain was decreased (in 12000 ppm females during lactation). At necropsy three of ten 12000 ppm (P) females exhibited a discolored, pale liver. There were increased absolute and relative weights of the adrenals, liver and kidneys (females) as well as the (both sexes) at 12000 ppm. At 12000 ppm decreased pup weights were observed from day 7 post partum onwards resulting in reduced absolute weights of the uterus. Therefore, the 2-generation reproduction study was dosed at 480, 2400 and 12000 ppm.

Positive control:

None

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily and once daily on weekends and holidays.
- Cage side observations included: All signs of illness or clinical reactions to treatment (especially during littering) were individually recorded daily. All further clinical symptoms were also recorded. This investigation included the observation of the general state of health, behavior, condition of the fur, and the orifices as well as excretory products. Any further findings e.g. prolonged parturition, morbidity and mortality were recorded as well.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Prior to the first administration in the diet and then weekly as a rule and during the pregnancy and lactation periods as follows:
- Pregnancy on Day 0 (= day of a sperm positive smear or vaginal plug) 7, 14 and 20.
- Lactation on Day 0 (day of birth), 4, 1, 14, 21 and 28.

BODY WEIGHT: Yes
- Time schedule for examinations: Body weights were recorded directly prior to the first administration and thereafter weekly up to necropsy (males and females not pregnant) and during the pregnancy and lactation periods as follows: During pregnancy on Day p.c. 0, 7, 14 and 20; During lactation on Day p.p. 0, 4, 7, 14, 21 and 28; On the day of scheduled necropsy.

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
The individual food consumption was measured (by weighing the quantity of food provided and back-weighing the amount, which remained unconsumed) as follows:
- Males: Weekly from Week 1 up to necropsy (except during mating period).
- Females: Weekly from Week 1 up to mating.
During pregnancy Day post coitum (p.c.) 0-7; 7-14; 14-20.
During lactation Day post partum (p.p.) 0-4, 4-7.
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.

OTHER:
Determination of insemination
During the mating periods vaginal smears were taken in the morning after rats had been co-housed over night to determine time of insemination and gestation length. The vaginal smears were obtained using a flame-sterilized platinum loop and plated out on slides. Smears were stained for about 1 minute with MAY GRUENWALD'S Eosine-methylene blue solution modified for microscopy. The date where sperms or a vaginal plug were found microscopically was taken as gestation Day 0 for calculating the gestation length. Females which exhibited marked weight gains although insemination had not been established were not further co-housed. No duration of pregnancy could be determined for these animals.

Oestrous cyclicity (parental animals):

Oestrus cycle length determination was done by evaluation of vaginal smears received daily over 19 consecutive days about 2 weeks prior to the mating period. The smears were examined microscopically for large serrated cells indicating that oestrus had occurred. This data was used to determine the oestrus cycle length and whether females were cycling properly.

Sperm parameters (parental animals):

Spermatological investigations were performed in all surviving (P) and (F1) males of the 0 and 12000 ppm group on the day of necropsy: Spermatozoa motilily, viability and morphology; Determination of spermatozoa in epididymis; Determination of homogenisation resistant spermatid heads in the testis

Litter observations:

STANDARDISATION OF LITTERS
- Performed on Day 4 postpartum: Yes
- Maximum of 8 pups/litter (4/sex/litter); excess pups (dead/alive) were examined for external effects.

PARAMETERS EXAMINED
The following parameters were examined in F1 / F2 offspring:
Number of live pups, pup weight, external alterations (=clinical and necropsy observation), number of dead pups, ano-genital measurements, sex of each pup, developmental milestones, including age and body weight when balano-preputial separation or vaginal opening occured.

GROSS EXAMINATION OF DEAD PUPS:
Yes, for external and internal abnormalities.

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.
All parental rats to be necropsied unscheduled were also killed by exsanguination under CO2 narcosis.

GROSS NECROPSY
- Gross necropsy was done on all rats and consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera. Implantation sites were counted after staining with 10% aqueous ammonium sulfide. In cases where implausible discrepancies existed between the number of observed implantation sites and the number of pups delivered (more pups than implantation sites) the number of pups was used as number of implantation sites. In case of autolytic rats or rats killed pre-scheduled the number of pups was taken as the number of implantation sites.
Vaginal smears were performed and evaluated at scheduled necropsy as precaution but were not reported. Rats found dead or which had to be killed in moribund condition were necropsied as early as possible in the same way.

HISTOPATHOLOGY / ORGAN WEIGHTS
The following tissues of adult (P) and (F1) rats were fixed in 10% formalin with the exception of total kidneys (P) or the left and two thirds of the right kidney and testes, which were fixed in Davidson's solution: Abnormalities, adrenals*, brain*, epididymides*, esophagus, kidneys*, larynx, liver*, ovaries and oviducts*, pituitary gland*, prostate*, physical identifier, seminal vesicles and coagulating glands*, skin in mammary region, spleen*, testes*, thyroid/parathyroids*, trachea, ureters, urinary bladder, uterus (incl. cervix)*, vagina, head with scull cap (* organ was weighed).
The organs mentioned above were microscopically examined at least in the control and high dose group. This included also all P/F1 rats, which died intercurrently (as far as possible) and those, which were killed moribund. Organs altered possibly by the treatment and any gross and/or microscopically changed lesions were examined in all rats. Staging of ovarian follicles was done in F1 females (control and high concentration) only. All reproductive organs of pairs without pups were investigated.

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring not selected as parental animals and all F2 offspring were sacrificed at 28 days of age.

GROSS NECROPSY
All pups culled for standardisation of litter size, found dead or had to be killed moribund during lactation were necropsied and investigated macroscopically with particular attention on the organs of reproduction except for cases of autolysis or cannibalism. This included also visible skeletal abnormalities as far as possible. A lung flotation in water was performed during the necropsy of pups found dead on the day of the first litter inspection to determine whether pups had breathed at birth (life birth) or not.
All F1 weanlings not selected for further treatment were killed when they were at least 4 weeks old and examined macroscopically.
All F2 weanlings were killed after a 4 week lactation period and examined macroscopically.

HISTOPATHOLOGY / ORGAN WEIGTHS
Grossly abnormal tissues if any were fixed in all pups/weanlings. In F1 and F2 weanlings brain, spleen, thymus, uterus, urinary bladder (instillation fixed), ureters, urethra and kidneys of one male and one female out of the firstly necropsied 5 litters per group were fixed in 10% formalin.
The brain, spleen, thymus and uterus of one male and one female per F1/F2 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:

Statistical evaluation was performed by a TASC-application (Toxicology Analysis Systems Customized) using the following methods:
Analysis of Variance (ANOVA) and in case of significant results Dunnett's test as post hoc test: Body weights/body weight gains of males/females, food consumption of the males and females, number of implantation sites/female, number of viable pups/female, organ weights at necropsy, time to insemination, life birth, viability and lactation index, oestrus data.
Chi-test and in case of significant differences Fisher's exact test with Bonferroni correction: Number of viable pups/group based on the number of implantations, insemination, fertility, gestation and rearing rate.
Kruskall-Wallis test and in case of significant differences Dunnett's test: number of prenatal loss per litter.

Sperm and spermatid count data were not evaluated statistically, because there were no meaningful differences between high dose and control groups.
Generally, differences between control group and treatment-groups were considered as statistically significant when p<0.05 (*) or p<0.01 (**).

Follicle counts were evaluated statistically using Wilcoxon Mann-Whitney U-test. Females without implantation sites and females and males, which died or which had to be sacrificed in moribund condition, were excluded from statistical evaluation of post mating parameters. This was true also for those (P) females, where additionally mated. Animals which spilled their food, if any, were excluded from calculation of means for food intake at the respective days.
Gestation length was not calculated for remated females with pups. Nutritional state of animals examined at their necropsy was reported only in cases where a deviation from normal nutritional state was noted. Mean pup weight of each individual litter was used as basis for calculating mean pup weight of the dose groups. Mean litter size calculation was based on number of female animals with living pups.

Reproductive indices:

The following reproductive indices were calculated from breeding and parturition records of animals in the study using the following formulae:
Insemination index (%): (No. of sperm positive females/No. of females co-housed with a male) x 100
Fertility index (%): (No. 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 p.p./No. of females that delivered a litter) x 100

Offspring viability indices:

The following viability indices were calculated from lactation records of litters in the study:
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 (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:

effects observed, treatment-related

Description (incidence and severity):

12000 ppm:
- Increase in the number of P females showing adverse clinical signs, including piloerection, poor general condition, paleness and emaciation. Tabulated data is presented in Attachment 1.

Dermal irritation (if dermal study):

not examined

Description (incidence and severity):

Not applicable.

Mortality:

mortality observed, treatment-related

Description (incidence):

12000 ppm:
- During lactation five P females died or had to be sacrificed moribund. Tabulated data is presented in Attachment 1.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

12000 ppm:
- During gestation and lactation P females gained less (p<0.01) body weight than controls. Tabulated data is presented in Attachment 2.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Compound intake:
- Based on food consumption and body weight data, the doses expressed as mean daily mg test substance/kg body weight during the premating, gestation and lactation periods are presented in Attachment 3. The values for the P and F1 generation are considered to be representative for the test substance intake for the entire study.

Food efficiency:

not examined

Description (incidence and severity):

Not applicable.

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

not examined

Description (incidence and severity):

Not applicable.

Ophthalmological findings:

not examined

Description (incidence and severity):

Not applicable.

Haematological findings:

not examined

Description (incidence and severity):

Not applicable.

Clinical biochemistry findings:

not examined

Description (incidence and severity):

Not applicable.

Endocrine findings:

not examined

Description (incidence and severity):

Not applicable.

Urinalysis findings:

not examined

Description (incidence and severity):

Not applicable.

Behaviour (functional findings):

not examined

Description (incidence and severity):

Not applicable.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Not applicable.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

There were no histopathological lesions induced by the test substance at 480 ppm. Incidences of treatment-related organ changes found from 2400 ppm onwards are summarised in Attachment 7.

Intercurrent mortality:
- The increased intercurrent mortality (incidence P:0/0/0/5), seen in 12000 ppm P females is secondary to induced renal lesions caused by precipitation of foreign material in the kidneys and lower urinary tract.

Reproductive tract:
- There was no evidence of any induced finding in the testes, epididymides, prostate and seminal vesicles/coagulating glands up to and including 12000 ppm.
- In the ovaries, the amount and size of corpora lutea was similar throughout the groups up to and including 12000 ppm.
- At 12000 ppm there was an increased incidence of corpora lutea with a foamy cytoplasm.
- In the uterus of 12000 ppm P females with foamy corpus luteum cells more unregressed implantation sites were found.
- The vagina of the P females affected by the changes in the corpora lutea and uterine implantation sites showed a highly mucified vaginal epithelium more often.
- The numbers of animals without progeny did not show any dose-correlation. The histopathological investigation of these rats showed no evidence of a test substance induced effect.

Urinary tract:
- In the kidneys of 12000 ppm rats, precipitates of foreign (crystalline) material - presumably the test compound or metabolite - could be found more frequently in the collecting ducts and renal pelvis than at 0 ppm. As sequels of this condition the following changes were observed in the kidneys of male and female parental rats at 2400 ppm (partly) and 12000 ppm:
- Diffuse tubular basophllia, intratubular granulocytes, papillary necrosis, medullary fibrosis, dilation of the renal pelvis and transitional cell hyperplasia.
- A slight elevation of ureteric hypertrophy was encountered also in 12000 ppm P females.
- A slightly elevated incidence of diffuse transitional cell hyperplasia was observed in the urinary bladder of 12000 ppm P females.

Adrenals, liver and spleen:
- In the adrenal glands of 12000 ppm P females, an increased incidence of hypertrophy of the zona glomerulosa was seen. This was considered to be induced by the renal findings such as foreign material, basophilic tubules and renal pelvic dilation, therefore, it was regarded as treatment-related.
- In the liver an increased incidence of condensed cytoplasm, which is the morphological correlate of a decreased glycogen content, was found in P females at 12000 ppm more frequently than at 0 ppm.
- In the spleen a comprised congestion, increased haemopoiesis and high grade of iron pigment deposits were observed more frequently in males (from 2400 ppm onwards) and females (at 12000 ppm) of the P generation indicating an increased turnover of red blood cells.

Histopathological findings: neoplastic:

not examined

Description (incidence and severity):

Not applicable.

Other effects:

not examined

Description (incidence and severity):

Not applicable.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

effects observed, treatment-related

Description (incidence and severity):

12000 ppm:
- A reduced rearing index was calculated for P females as result of increased mortality of 12000 ppm dams (5 deaths) and some total litter losses of females. A summary of reproductive performance is presented in Attachment 4.

Effect levels (P0)

1

Key result

Dose descriptor:

NOAEL

Remarks:

systemic

Effect level:

480 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects at this dose level.

Remarks on result:

other: corresponding to 39 and 55 mg/kg bw/day for males and females, respectively.

2

Key result

Dose descriptor:

LOAEL

Remarks:

systemic

Effect level:

2 400 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

organ weights and organ / body weight ratios

histopathology: non-neoplastic

Remarks on result:

other: corresponding to 202 and 260 mg/kg bw/day for males and females, respectively.

3

Key result

Dose descriptor:

NOAEL

Remarks:

reproductive

Effect level:

2 400 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects at this dose level.

Remarks on result:

other: corresponding to 173 mg/kg bw/day.

4

Key result

Dose descriptor:

LOAEL

Remarks:

reproductive

Effect level:

12 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reproductive performance

Remarks on result:

other: corresponding to 840 mg/kg bw/day.

Target system / organ toxicity (P0)

1

Critical effects observed:

yes

Lowest effective dose / conc.:

2 400 ppm

System:

urinary

Organ:

kidney

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

no

2

Critical effects observed:

yes

Lowest effective dose / conc.:

2 400 ppm

System:

haematopoietic

Organ:

spleen

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Results: P1 (second parental generation)

General toxicity (P1)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

12000 ppm:
- There was an increase in number of F1 male and female rats showing clinical signs such as poor general condition, piloerection, paleness, high-stepping gate, sunken flanks and/or, emaciation. Tabulated data is presented in Attachment 1.

Dermal irritation (if dermal study):

not examined

Description (incidence and severity):

Not applicable.

Mortality:

mortality observed, treatment-related

Description (incidence):

12000 ppm:
- Four F1 females were sacrificed pre-scheduled, two of them died and two had to be sacrificed moribund.

480 ppm:
- One F1 female had to be killed moribund.

Control:
- One F1 male died after mating.

Tabulated data is presented in Attachment 1.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

12000 ppm:
- At termination of the premating period, F1 females exhibited 9% lower (p<0.01) body weights than controls. There was also a slight body weight depression (-4.5% compared to controls) in F1 males receiving 12000 ppm, which was not statistically significant.
- During gestation and lactation F1 females gained less (p<0.01) body weight than controls.

Tabulated data is presented in Attachment 2.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Compound intake:
- Based on food consumption and body weight data, the doses expressed as mean daily mg test substance/kg body weight during the premating, gestation and lactation periods are presented in Attachment 3. The values for the F0 and F1 generation are considered to be representative for the test substance intake for the entire study.

Food efficiency:

not examined

Description (incidence and severity):

Not applicable.

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

not examined

Description (incidence and severity):

Not applicable.

Ophthalmological findings:

not examined

Description (incidence and severity):

Not applicable.

Haematological findings:

not examined

Description (incidence and severity):

Not applicable.

Clinical biochemistry findings:

not examined

Description (incidence and severity):

Not applicable.

Endocrine findings:

not examined

Description (incidence and severity):

Not applicable.

Urinalysis findings:

not examined

Description (incidence and severity):

Not applicable.

Behaviour (functional findings):

not examined

Description (incidence and severity):

Not applicable.

Immunological findings:

not examined

Description (incidence and severity):

Not applicable.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Organ measurements revealed a tendency to increased absolute and relative spleen weights in F1 males and females from 2400 ppm onwards, which were partly statistically significant. There was also a tendency to increased absolute and relative thyroid weights in F1 females from 2400 ppm onwards. Relative kidney weights were statistically significantly increased in F1 females at 12000 ppm. Other statistical significances or weight deviations were not toxicologically significant as a dose correlation was missing, the differences were minor or caused by variations in body weights. A summary of absolute and relative organ weights for F1 parental animals is presented in Attachment 6.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

12000 ppm:
- 7/25 F1 females were found to be skinny.
- An increased incidence of gross changes in the kidneys was found F1 females (incidences with increasing dose: discoloration 1/0/0/5, enlargement 0/0/0/2, surface changes 1/0/0/5).

Neuropathological findings:

not examined

Description (incidence and severity):

Not applicable.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Intercurrent mortality:
- The increased intercurrent mortality (incidence F1: 0/1/0/4) seen in 12000 ppm F1 females was secondary to induced renal lesions caused by precipitation of foreign material in the kidneys and lower urinary tract.
- Female No. 332 (480 ppm, F1 generation) exhibited an intrauterine fetus and fluid, which corresponded histopathologically to marked metritis and perimetritis.

Reproductive tract:
- There was no evidence of any induced finding in the testes, epididymides, prostate and seminal vesicles/coagulating glands up to and including 12000 ppm.
- In the ovaries, the amount and size of corpora lutea was similar throughout the groups up to and including 12000 ppm.
- At 12000 ppm there was an increased incidence of corpora lutea with a foamy cytoplasm.
- Counting of primary follicles and corpora lutea done in controls and 12000 ppm F1 females revealed no treatment-related changes.
- The numbers of animals without progeny did not show any dose-correlation.
- The histopathological investigation of these rats showed no evidence of a test substance induced effect.

Urinary tract:
- In the kidneys of 12000 ppm rats precipitates of foreign (crystalline) material - presumably the test compound or metabolite - could be found more frequently in the collecting ducts and renal pelvis than at 0 ppm. As sequels of this condition the following changes were observed in the kidneys of male and female parental rats at 2400 ppm (partly) and 12000 ppm: Diffuse tubular basophllia, intratubular granulocytes, papillary necrosis, medullary fibrosis, dilation of the renal pelvis and transitional cell hyperplasia. In 12000 ppm females in addition cortical tubules showed small vacuoles or inclusions, which were more frequent in the F1 generation.
- In 12000 ppm F1 females the ureters showed an increased incidence of hypertrophy. A slight elevation of ureteric hypertrophy was also seen at 2400 ppm.
- A slightly elevated incidence of diffuse transitional cell hyperplasia was observed in the urinary bladder of 12000 ppm F1 females.

Adrenals, liver and spleen:
- In the adrenal glands of 12000 ppm F1 females and one F1 male, an increased incidence of hypertrophy of the zona glomerulosa was seen. This was considered to be induced by the renal findings such as foreign material, basophilic tubules and renal pelvic dilation, therefore, it was regarded as treatment-related.
- In the liver an increased incidence of condensed cytoplasm, which is the morphological correlate of a decreased glycogen content, was found in F1 females at 12000 ppm more frequently than at 0 ppm.
- In the spleen a comprised congestion, increased haemopoiesis and high grade of iron pigment deposits were observed more frequently in males (partly from 2400 ppm onwards) and females (at 12000 ppm) of the F1 generation indicating an increased turnover of red blood cells.

Histopathological findings: neoplastic:

not examined

Description (incidence and severity):

Not applicable.

Other effects:

not examined

Description (incidence and severity):

Not applicable.

Reproductive function / performance (P1)

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

Effect levels (P1)

1

Key result

Dose descriptor:

NOAEL

Remarks:

systemic

Effect level:

480 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects at this dose level.

Remarks on result:

other: corresponding to 50 and 69 mg/kg bw/day for males and females, respectively.

2

Key result

Dose descriptor:

LOAEL

Remarks:

Systemic

Effect level:

2 400 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

organ weights and organ / body weight ratios

histopathology: non-neoplastic

Remarks on result:

other: corresponding to 252 and 324 mg/kg bw/day for males and females, respectively.

3

Key result

Dose descriptor:

NOAEL

Remarks:

reproductive

Effect level:

12 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects at this dose level.

Remarks on result:

other: corresponding to 1292 and 1001 mg/kg bw/day for males and females, respectively.

Target system / organ toxicity (P1)

1

Critical effects observed:

yes

Lowest effective dose / conc.:

2 400 ppm

System:

urinary

Organ:

kidney

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

no

2

Critical effects observed:

yes

Lowest effective dose / conc.:

2 400 ppm

System:

haematopoietic

Organ:

spleen

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Results: F1 generation

General toxicity (F1)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

12000 ppm:
- Incidences of pups exhibiting the clinical findings of no milk spot, paleness thin and/or cold to touch were increased.

A summary of clinical findings and litter parameters of F1 and F2 generations are presented in attachment 8.

Dermal irritation (if dermal study):

not examined

Description (incidence and severity):

Not applicable.

Mortality / viability:

mortality observed, treatment-related

Description (incidence and severity):

12000 ppm:
- F1 pups exhibited a reduced (p>0.05) viability index (litters of dams, which died during lactation were excluded) as a result of an increase in pup deaths (including total litter loss in 2 females), which must be interpreted as a treatment-related effect though a comparable low viability index in control F2 pups was noted and no effect was seen on viability of 12000 ppm F2 pups.
- The lactation indices were slightly reduced in both generations. Though the lactation indices at 12000 ppm were within historical control range a test substance-related effect on this parameter could not be excluded, if the toxicity observed in the dams is considered.

The fact that there were higher mean litter sizes of F2 pups in group 2400 and 12000 ppm than in F1 pups of corresponding dose groups is based on the result that some F1 litters had died completely.

A summary of clinical findings and litter parameters of F1 and F2 generations are presented in attachment 8.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

12000 ppm:
- Treatment-related depressed pup (males and females) and litter weights were evident (pups: partly statistically significant from Day 7 p.p. onwards); litters: about 11-16% from Day 0 p.p. onwards).

A summary of mean litter and pup weights is presented in Attachment 9.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Compound intake:
- Based on food consumption and body weight data, the doses expressed as mean daily mg test substance/kg body weight during the premating, gestation and lactation periods are presented in Attachment 3. The values for the P and F1 generation are considered to be representative for the test substance intake for the entire study.

Food efficiency:

not examined

Description (incidence and severity):

Not applicable.

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

not examined

Description (incidence and severity):

Not applicable.

Ophthalmological findings:

not examined

Description (incidence and severity):

Not applicable.

Haematological findings:

not examined

Description (incidence and severity):

Not applicable.

Clinical biochemistry findings:

not examined

Description (incidence and severity):

Not applicable.

Urinalysis findings:

not examined

Description (incidence and severity):

Not applicable.

Sexual maturation:

effects observed, treatment-related

Description (incidence and severity):

12000 ppm:
- Female F1 post weanlings exhibited a slight delay in time of vaginal opening (p<0.01). This finding was interpreted to be due to body weight retardation. Additionally, an estrogenic or androgen effect was excluded as ano-genital measurements in F2 pups revealed no differences between controls and treated animals up to 12000 ppm.

Summary data of sexual maturation milestones for F1 post-weanlings is presented in Attachment 10.

Anogenital distance (AGD):

not examined

Description (incidence and severity):

Not applicable.

Nipple retention in male pups:

not examined

Description (incidence and severity):

Not applicable.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

12000 ppm:
- For some absolute or relative organ weights statistically significantly higher or lower means were obtained compared to means of the control group. In all cases, these changes were attributed to differences in body weight as follows:
- Reduced absolute thymus weights in female F1 weanlings were not correlated with statistically significantly changed relative weights.
- Elevated relative brain weights of F1 females corresponded with lower absolute brain weights.

A summary of organ weight data for F1 weanlings is presented in Attachment 11.

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

Description (incidence and severity):

Not applicable.

Other effects:

not examined

Description (incidence and severity):

Not applicable.

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Description (incidence and severity):

Not applicable.

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Description (incidence and severity):

Not applicable.

Effect levels (F1)

1

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

2 400 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects at this dose level.

Remarks on result:

other: corresponding to 173 mg/kg bw/day.

2

Key result

Dose descriptor:

LOAEL

Generation:

F1

Effect level:

12 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

viability

clinical signs

body weight and weight gain

Remarks on result:

other: corresponding to 841 mg/kg bw/day.

Target system / organ toxicity (F1)

Critical effects observed:

no

Results: F2 generation

General toxicity (F2)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

12000 ppm:
- The incidences of pups exhibiting the clinical findings of no milk spot, paleness thin and/or cold to touch were increased by the intake of the test substance.

A summary of clinical findings and litter parameters of F1 and F2 generations are presented in attachment 8.

Dermal irritation (if dermal study):

not examined

Description (incidence and severity):

Not applicable.

Mortality / viability:

no mortality observed

Description (incidence and severity):

The lactation indices were not changed remarkably up to 2400 ppm and slightly reduced at 12000 ppm in both generations. Though the lactation indices at 12000 ppm were within historical control range a test substance-related effect on this parameter can not be excluded, if the toxicity observed in the dams is considered.

A summary of clinical findings and litter parameters of F1 and F2 generations are presented in attachment 8.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

12000 ppm:
- Treatment-related depressed pup (males and females) and litter weights were evident (pups: partly statistically significant from Day 7 p.p. onwards); litters: about 11-16% from Day 0 p.p. onwards).

A summary of mean litter and pup weights is presented in Attachment 9.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Compound intake:
- Based on food consumption and body weight data, the doses expressed as mean daily mg test substance/kg body weight during the premating, gestation and lactation periods are presented in Attachment 3. The values for the F1 and F2 generation are considered to be representative for the test substance intake for the entire study.

Food efficiency:

not examined

Description (incidence and severity):

Not applicable.

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

not examined

Description (incidence and severity):

Not applicable.

Ophthalmological findings:

not examined

Description (incidence and severity):

Not applicable.

Haematological findings:

not examined

Description (incidence and severity):

Not applicable.

Clinical biochemistry findings:

not examined

Description (incidence and severity):

Not applicable.

Urinalysis findings:

not examined

Description (incidence and severity):

Not applicable.

Sexual maturation:

not examined

Anogenital distance (AGD):

no effects observed

Nipple retention in male pups:

not examined

Description (incidence and severity):

Not applicable.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

12000 ppm:
- For some absolute or relative organ weights statistically significantly higher or lower means were obtained compared to means of the control group. In all cases, these changes were attributed to differences in body weight as follows:
- Reduced absolute thymus weights in F2 weanlings were not correlated with statistically significantly changed relative weights.
- Reduced absolute spleen weights of male F2 weanlings were not associated with statistically significant changed relative weights.
- Elevated relative brain weights of F2 weanlings corresponded with lower absolute brain weights.

A summary of organ weight data for F2 weanlings is presented in Attachment 11.

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

Description (incidence and severity):

Not applicable.

Other effects:

not examined

Description (incidence and severity):

Not applicable.

Developmental neurotoxicity (F2)

Behaviour (functional findings):

not examined

Description (incidence and severity):

Not applicable.

Developmental immunotoxicity (F2)

Developmental immunotoxicity:

not examined

Description (incidence and severity):

Not applicable.

Effect levels (F2)

1

Key result

Dose descriptor:

NOAEL

Generation:

F2

Effect level:

2 400 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects at this dose level.

Remarks on result:

other: corresponding to 196 mg/kg bw/day.

2

Key result

Dose descriptor:

LOAEL

Generation:

F2

Effect level:

12 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

viability

clinical signs

body weight and weight gain

Remarks on result:

other: corresponding to 1001 mg/kg bw/day.

Target system / organ toxicity (F2)

Critical effects observed:

no

Overall reproductive toxicity

Key result

Reproductive effects observed:

yes

Lowest effective dose / conc.:

12 000 ppm

Treatment related:

yes

Relation to other toxic effects:

reproductive effects as a secondary non-specific consequence of other toxic effects

Dose response relationship:

yes

Relevant for humans:

not specified

Applicant's summary and conclusion

Conclusions:

The study was performed in accordance to OECD TG 416 under GLP conditions and is considered reliable. Under the conditions described, the parental systemic LOAEL in rats is 2400 ppm (corresponding to 202 mg/kg bw/day in males and 260 mg/kg bw/day in females) and the parental systemic NOAEL is 480 ppm (39 mg/kg bw/day in males and 55 mg/kg bw/day in females), based on histopathological changes in spleen and urinary tract accompanied with organ weight changes. The NOAEL for parental reproductive parameters in the P animals was 2400 ppm (173 mg/kg bw/day) due to a reduced rearing index at 12000 ppm. Parental animals of the F1 generation did not show any adverse effect on reproductive behaviour or function, and thus, a NOAEL of 12000 ppm was derived for the F1 parental animals. The offspring NOAEL is 2400 ppm (173 mg/kg bw/day in F1 offspring and 196 mg/kg bw/day in F2 offspring) due to pup weight decrease at 12000 ppm.