clothianidin (ISO); (E)-1-(2-chloro-1,3-thiazol-5-ylmethyl)-3-methyl-2-nitroguanidine

Toxicity to reproduction

Administrative data

Endpoint:

two-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

29 Jun 1998 - 6 Apr 1999

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference 1

Reference Type:

study report

Title:

Unnamed

Year:

2000

Report date:

2000

Reference 2

Reference Type:

other: Amendment

Title:

Unnamed

Year:

2001

Report date:

2001

Reference 3

Reference Type:

other: Amendment

Title:

Unnamed

Year:

2003

Report date:

2003

Reference 4

Reference Type:

other company data

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)

Version / remarks:

adopted 2001

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)

Version / remarks:

adopted 1983

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

433-460-1

EC Name:

-

Cas Number:

210880-92-5

Molecular formula:

C6H8ClN5O2S

IUPAC Name:

(E)-N'-[(2-chloro-1,3-thiazol-5-yl)methyl]-N-methyl-N''-nitroguanidine

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Raleigh, NC
- Females nulliparous and non-pregnant: yes
- Age at study initiation: (P) 7 - 8 wks; (F1) 3 wks
- Weight at study initiation: (P) Males: 261.0 - 272.3 g; Females: 185.1 - 188.6 g
- Housing: individually (except during mating phase) in suspended stainless steel cages
- Diet: Purina Mills Rodent Lab Chow 5001-4, ad libitum
- Water: municipal tap water, ad libitum
- Acclimation period: at least six days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 - 26
- Humidity (%): 30 - 70
- Photoperiod (hrs dark / hrs light): 12 / 12

IN-LIFE DATES: From: 26 Jun 1988 To: 6 Apr 1999

Administration / exposure

Route of administration:

oral: feed

Details on exposure:

DIET PREPARATION
- Rate of preparation of diet: weekly
- Mixing appropriate amounts with: Purina Mills Rodent Lab Chow 5001-4 and corn oil as carrier for the compound and to control dust formation
- Storage temperature of food: under refrigerator conditions

Details on mating procedure:

- M/F ratio per cage: 1/1
- Length of cohabitation: up to 14 consecutive days
- Proof of pregnancy: vaginal plug and/or sperm in vaginal smear (daily examination); referred to as Day 0 of gestation

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The homogeneity and stability of the test substance in rodent feed was verified prior to initiation of the study. Feed samples were taken from each dietary level on study weeks 1, 2, 3 and at monthly intervals thereafter. Samples were maintained at refrigerator conditions prior to analysis. The test item was found to be homogenously distributed since the coefficient of variation (CV) of the samples is less than 10%. The test item was also found to be stable in feed for up to 14 days (room temperature). The mean analytical determined dietary concentrations of test item found in the diet were 91, 88, and 89% of nominal concentrations for the 150, 500, and 2500 ppm dose groups, respectively.

Frequency of treatment:

continously via the diet

Details on study schedule:

Selection of parents from F1 generation when pups were 21 days of age.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

150 ppm

Remarks:

Corresponding to the actual dose ingested during the premating phases of both generations combined: 10.2 mg/kg bw/day (males), 11.8 mg/kg bw/day (females)

Doses / concentrations 2

Dose / conc.:

500 ppm

Remarks:

Corresponding to the actual dose ingested during the premating phases of both generations combined: 32.7 mg/kg bw/day (males), 37.9 mg/kg bw/day (females)

Doses / concentrations 3

Dose / conc.:

2 500 ppm

Remarks:

Corresponding to the actual dose ingested during the premating phases of both generations combined: 179.6 mg/kg bw/day (males), 212.9 mg/kg bw/day (females)

No. of animals per sex per dose:

(P0): 30
(P1): 30

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: Dose selection was performed on basis of results obtained in previous studies (chronic toxicity study and pilot reproduction toxicity study). The selected dose range was expected to produce evidence of toxicity at the highest dietary concentration and no parental or reproductive effects at the lowest dietary concentration.

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily, once a day during weekends and holidays

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once a week and if an animal showed clinical signs during daily cage side observations

BODY WEIGHT: Yes
- Time schedule for examinations: once a week during premating (males + females), once per week during mating period and until sacrifice (males), during gestation body weight of dams was measured on Days 0, 6, 13 and 20, during lactation dam body weight was measured on Days 0, 4, 7, 14 and 21.

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

Oestrous cyclicity (parental animals):

Oestrous cycle was characterized for all females by examining daily vaginal smears over a three-week period prior to mating and just prior to termination.

Sperm parameters (parental animals):

For all P0 and P1 male parental animals, sperm was collected from one testes and one epididymis for enumeration of homogenization-resistant spermatids and cauda epididymal sperm reserves, respectively. Additionally, sperm motility and sperm morphology were evaluated from the distal portion of the vas deferens.

Litter observations:

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

PARAMETERS EXAMINED
The following parameters were examined in F1 and F2 offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities, anogenital distance (AGD) (only F2 on lactation Day 0).

Further, in F1 offspring grown to adults also the following parameters were examined:
clinical signs, body weights, vaginal opening, preputial separation and food consumption.

GROSS EXAMINATION OF DEAD PUPS:
yes, gross necropsy was performed born or found dead

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals following the mating phase
- Maternal animals: All surviving animals following the weaning of their respective litters
-Female animals that were sperm positive and/or had an internal vaginal plug but did not deliver were sacrificed after GD 24. Females which were never observed as being inseminated and/or with an internal vaginal plug were sacrificed at least 24 days after the completion of the mating phase, if they did not deliver. In these animals in addition to gross necropsy an evaluation of patency of the cervical/uterine os was performed.

GROSS NECROPSY
- Gross necropsy consisted of external and internal examination. The uterus was excised and the implantation sites were counted.

HISTOPATHOLOGY / ORGAN WEIGHTS
Ovaries, uterus, brain, pituitary, thymus, liver, kidneys, adrenal glands, spleen, testes, epididymides, seminal vesicles and prostate were removed and weighed. Microscopical examination: cervix, epididymis, gross lesions, adrenals, liver, ovaries, pituitary, prostate, testicles, seminal vesicles/coagulating gland, uterus, and vagina. Histopathological evaluations were conducted on control and top dose group, except for reproductive organs which were analysed for all animals demonstrating affected fertility (failed to mate, conceive, sire, or deliver healthy offspring).

Furthermore, a quantitative evaluation of the primordial ovarian follicles was conducted in F1 dams if any of the following were ascribed to treatment: 1) a reduction in ovarian weight and abnormal ovarian histopathology findings, 2) abnormal oestrous cyclicity and female infertility, and/or 3) depletion is testicular spermatid counts in the F1 males and evidence of germ cell depletion during testicular histopathology evaluations.

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring not selected as parental animals and all F2 offspring were sacrificed at 21 days of age.
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows:

GROSS NECROPSY
- Gross necropsy consisted of macroscopical examination for any structural abnormalities or pathological changes, particularly as they may relate to the organs of the reproductive system
-Pups found dead were examined for possible defects and/or cause of death

HISTOPATHOLOGY / ORGAN WEIGTHS
Additionally the brain, spleen, and thymus were weighed from one randomly selected pup/sex/litter in those pups examined macroscopically

Statistics:

Parametric data (including body weight gain and food consumption) were analyzed using a univariate Analysis of Variance (ANOVA), and if significant differences were observed a Dunnett's Test was performed. Nonparametric data (e.g., number of oestrous cycles, litter site, and number of implantation sites) were first analyzed by the Kruskal-Wallis test and then subjected to Dunn's Test if significant differences were identified. Nonparametric dichotomous data (e.g. fertility and gestation indices) were initially analyzed by the Chi-Square Test and if significance was observed between groups then by the Fisher's Exact Test with the Bonferroni adjustment. To the extent possible, the frequency of gross lesions were first examined visually, then, in the event of questionable distribution, by statistical analysis using the Chi-Square and Fisher's exact tests. Comparisons were made at both the 0.05 and 0.01 levels of significance. Sperm morphology and count were analyzed in the following way: The homogeneity of variances was tested with the folded form of the F statistic at the 0.01 significance level. If variances were not significantly different, the high dose group was compared with the control with the Cochran and Cox t-test. Animals with less than 100 available sperm were not included in the analysis of sperm morphology. For sperm motility, a trend test was conducted in SAS PROC MULTTEST using the ordinal dose scale and the permutation adjusted p-value. If the trend test with 4 groups was significant (p <= 0.05), the high dose group was deemed significantly different from control and the trend test was repeated with the 3 remaining groups (control, low and mid dose). The analysis process continued until the trend test was not statistically significant.

Reproductive indices:

Mating index (%) = (number of inseminated females / number of females co-housed) * 100

Fertility index (%) = (number of pregnant females / number of inseminated females) * 100

Gestation index (%) = (number of females with live pups / number of pregnant females) * 100

Birth index (%) = (total number of pups born per litter / total number of implantation sites per litter) * 100

Live birth index (%) = (number of pups born per litter / total number of pups per litter) * 100

Viability index (%) = (number of live pups per litter on day 4 (pre-culling) / number of live pups born per litter) * 100

Lactation index (%) = (number of live pups per litter on day 21 / number of live pups per litter on day 4 (post-culling)) * 100

-inseminated females include pregnant females not observed sperm positive or to have had an internal vaginal plug
-pregnant females include females which did not deliver, but had implantation sites

Offspring viability indices:

Viability index = (number of live pups/litter on Day 4 (pre-culling) / number of live pups born/litter) * 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Clinical signs such as lacrimation of eyes, hair thinning or chromorhinorrhea of the nose were only observed in individual cases and without any dose-dependency. They are considered to be of no toxicological relevance.

Dermal irritation (if dermal study):

not examined

Description (incidence and severity):

not applicable

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One female rat each of the control and 2500 ppm group died before sacrifice. Both deaths were considered to be incidental and not compound-related.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Statistically significant decreased body weights were observed in male and female rats of the 2500 ppm dose group from Day 7 onwards. Male body weights were decreased by 10 % compared to control on Day 126. Female body weights were decreased by 13% compared to control on Day 70. During gestation phase, also a decreased body weight was noted for females of the top dose group (-12 to -14% compared to control). During lactation period, body weights of dams were decreased at all time points (2500 ppm) or on lactation Day 14 (500 ppm), respectively. No effect on body weight or weight changes during any study period was noted in the 150 ppm group.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Food consumption was affected in male and female rats during the pre-mating phase in the 2500 ppm dose group. In the males, the food consumption was decreased during the first week and then increased afterwards for all subsequent time points, relative to control. In the females, food consumption was also decreased during the first week, then similar to control during weeks 2 and 3 and then increased for the remainder of pre-mating period. In the 500 ppm group a significantly lowered food consumption was observed during the first two weeks of pre-mating phase. The decrease in food consumption noted during the first weeks of a dietary study are not unusual and typically related to feed palatability, and as such are not considered to be toxicologically meaningful. During gestation phase, food consumption of females dosed with 2500 ppm test item was increased (+11 % compared to control) from days 6 to 13 of this phase. No effect on food consumption was noted during lactation phase.

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, non-treatment-related

Description (incidence and severity):

All histopathological lesions were considered to be incidental and not compund-related.

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

Description (incidence and severity):

No significant effects on either oestrous cycle length or number of cycles were observed. The term cycle stage data was not statistically analyzed for significant findings as there were no histological ovarian findings.

Reproductive function: sperm measures:

effects observed, treatment-related

Description (incidence and severity):

Sperm morphology and total sperm count were not affected in the top dose group when compared to control and thus not analyzed in the 150 and 500 ppm groups. A statistically significant decrease in sperm motility (percent progressively motile, but not percent motile) was observed in the 2500 ppm group. This effect is considered to be secondary to body weight changes also observed at this dose level. However, according to the RAC opinion, classification of the test substance for fertility and sexual function as Repr. 2 (H361f) is warranted mainly based on delayed male sexual maturation supported by alterations in sperm motility. No effects on sperm motility were noted in the 150 and 500 ppm groups. For details please refer to attachment 07.

Reproductive performance:

no effects observed

Description (incidence and severity):

The mating index was 90, 100, 100, and 100%, the fertility index was 92, 97, 93, and 97%, and the gestation index was 100, 100, 100, and 100% for the control, 150, 500 and 2500 ppm groups, respectively. There were no significant effects on days to insemination, gestation length, or the median number of implants.

Effect levels (P0)

1

Key result

Dose descriptor:

NOAEL

Remarks:

reproductive toxicity

Effect level:

500 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reproductive performance

other: no adverse effect observed

Remarks on result:

other: Corresponding to 32.7 mg/kg bw/d (males) and 37.9 mg/kg bw/d (females)

2

Key result

Dose descriptor:

LOAEL

Remarks:

reproductive toxicity

Effect level:

2 500 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reproductive performance

Remarks on result:

other: Corresponding to 179.6 mg/kg bw/d (males) and 212.9 mg/kg bw/d (females)

3

Key result

Dose descriptor:

NOAEL

Remarks:

general toxicity

Effect level:

150 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other:

Remarks on result:

other: Corresponding to 10.2 mg/kg bw/d (males) and 11.8 mg/kg bw/d (females)

4

Key result

Dose descriptor:

LOAEL

Remarks:

general toxicity

Effect level:

500 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

food consumption and compound intake

Remarks on result:

other: Corresponding to 32.7 mg/kg bw/d (males) and 37.9 mg/kg bw/d (females)

Target system / organ toxicity (P0)

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

2 500 ppm

System:

male reproductive system

Treatment related:

yes

Dose response relationship:

not specified

Relevant for humans:

not specified

Results: P1 (second parental generation)

General toxicity (P1)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Clinical signs such as lacrimation of eyes, hair thinning or chromorhinorrhea of the nose were only observed in individual cases and without any dose-dependency. They are considered to be of no toxicological relevance.

Dermal irritation (if dermal study):

not examined

Description (incidence and severity):

not applicable

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One female rat of the 150 ppm group was sacrificed unscheduled and exhibited a severe lung abscess with inhalation pneumonia. It was considered to be incidental and not compound-related.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Statistically significant decreased body weights were observed in male and female rats of the 2500 ppm dose group throughout pre-mating. Male body weights were decreased by 21 % compared to control on Day 0 and an 18% decrease on Day 119. Female body weights were decreased constantly by 16 - 17% compared to control throughout the pre-mating phase. During gestation phase, also a decreased body weight was noted for females of the top dose group (-13 to -16% compared to control). During lactation period, body weights of dams were decreased at all time points (2500 ppm). No effect on body weight or weight changes during any study period was noted in the 150 and 500 ppm groups.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Food consumption was significantly increased in male and female rats during the pre-mating phase in the 2500 ppm dose group. In the pre-mating phase this effect ranged from 10 - 27 % (males) and 10- 30 % (females) as compared to control. During gestation phase, food consumption of females dosed with 2500 ppm test item was increased (+11 % compared to control) from days 6 to 13 of this phase. Food consumption was significantly increased during the last two weeks of lactation phase in the top dose females. No effects on food consumption during any phase was observed for the 150 and 500 ppm groups.

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):

Significantly decreased organ weights in both males and females of the 2500 ppm group were found for the thymus, but considered an indirect effect related to decreased body weight noted for this dietary level. All other observations related to organ weight changes are considered incidental since no dose-response relationships were established. See attachment 06 for details.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

All gross necropsy observations were considered to be incidental and not compound-related.

Neuropathological findings:

not examined

Description (incidence and severity):

not applicable

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

All histopathological lesions were considered to be incidental and not compund-related.

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

Description (incidence and severity):

No significant effects on either oestrous cycle length or number of cycles were observed. The term cycle stage data was not statistically analyzed for significant findings as there were no histological ovarian findings.

Reproductive function: sperm measures:

effects observed, treatment-related

Description (incidence and severity):

Sperm morphology and total sperm count were not affected in the top dose group when compared to control and thus not analyzed in the 150 and 500 ppm groups. A statistically significant decrease in sperm motility (percent progressively motile and percent motile) was observed in the 2500 ppm group. No effects on sperm motility were noted in the 150 and 500 ppm groups.

Reproductive performance:

effects observed, non-treatment-related

Description (incidence and severity):

The mating index was 93, 97, 93, and 93%, the fertility index was 82, 89, 75, and 100%, and the gestation index was 100, 100, 95, and 100% for the control, 150, 500 and 2500 ppm groups, respectively. There were no significant effects on days to insemination, gestation length, or the median number of implants.

Effect levels (P1)

1

Key result

Dose descriptor:

NOAEL

Remarks:

reproductive toxicity

Effect level:

500 ppm

Based on:

test mat.

Sex:

male

Basis for effect level:

other:

Remarks on result:

other: Corresponding to 32.7 mg/kg bw/d

2

Key result

Dose descriptor:

LOAEL

Remarks:

reproductive toxicity

Effect level:

2 500 ppm

Based on:

test mat.

Sex:

male

Basis for effect level:

reproductive function (sperm measures)

Remarks on result:

other: Corresponding to 179.6 mg/kg bw/d

3

Key result

Dose descriptor:

NOAEL

Remarks:

general toxicity

Effect level:

500 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other:

Remarks on result:

other: Corresponding to 32.7 mg/kg bw/d (males) and 37.9 mg/kg bw/d (females)

4

Key result

Dose descriptor:

LOAEL

Remarks:

general toxicity

Effect level:

2 500 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

Remarks on result:

other: Corresponding to 179.6 mg/kg bw/d (males) and 212.9 mg/kg bw/d (females)

Target system / organ toxicity (P1)

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

2 500 ppm

System:

male reproductive system

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Results: F1 generation

General toxicity (F1)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Only in individual litters pups with clinical signs such as weakness or bruising were observed.

Dermal irritation (if dermal study):

not examined

Description (incidence and severity):

not applicable

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

An increased incidence (statistically not significant) of stillborn pups was noted in the 2500 ppm dose group (2.3 % overall incicdence). However, the live-birth index, which describes the relative number of live pups born versus the total number of pups born per litter was not affected. In reproductive toxicity studies conducted in the testing laboratory on the Sprague-Dawley rat over the past three years, the incidence of pup stillbirths in the control groups has typically ranged from 0 to 3.9%. Therefore, this effect is considered to be within normal variations.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

In the 2500 ppm group (both sexes), mean pup body weights were significantly decreased relative to control troughtout lactation, as well as pup body weight change. In the 500 ppm group, pup body weight change was significantly affected from lactation Day 7 to 14. These effects are considered secondary to general maternal toxicity. No changes in body weight or body weight change were observed in the 150 ppm group.

Food consumption and compound intake (if feeding study):

not examined

Description (incidence and severity):

not applicable (food consumption for F1 generation was measured beginning with 10-week premating phase; see P1 section above)

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):

Preputial separation was observed on days significantly different from control in the 500 and 2500 ppm groups, while no effect in the 150 ppm group was noted. Time to vaginal opening was affected in the 2500 ppm group compared to control, but not in either 150 or 500 ppm groups. The effects observed are considered a consequence of the reduced pup growth demonstrated by retarded body weights and body weight changes in these groups.

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):

In the 2500 ppm group organ weights in male and female pups were decreased for the brain, thymus and spleen (absolute weights). For the same animals the relative brain weight was increased, while relative thymus and spleen weights were decreased. At the 500 ppm dose level absolute thymic weights were significantly decreased in male pups. See attachment 06 for details.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Gross necropsy observations made for pups sacrificed after 21 days and found dead pups were considered to be incidental and not compound-related. However, it should be noted, that lungs that did not float were observed in the stillborn pups described above.

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:

LOAEL

Generation:

F1

Effect level:

500 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

viability

sexual maturation

body weight and weight gain

Remarks on result:

other: Corresponding to 32.7 mg/kg bw/d (males) and 37.9 mg/kg bw/d (females)

2

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

150 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other:

Remarks on result:

other: Corresponding to 10.2 mg/kg bw/d (males) and 11.8 mg/kg bw/d (females)

Target system / organ toxicity (F1)

Key result

Critical effects observed:

no

Lowest effective dose / conc.:

500 ppm

System:

male reproductive system

Organ:

not specified

Results: F2 generation

General toxicity (F2)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Only in individual litters pups with clinical signs such as weakness or bruising were observed.

Dermal irritation (if dermal study):

not examined

Description (incidence and severity):

not applicable

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

An increased incidence (statistically not significant) of stillborn pups was noted in the 2500 ppm dose group (3.7 % overall incidence). However, the live-birth index, which describes the relative number of live pups born versus the total number of pups born per litter was not affected. In reproductive toxicity studies conducted in the testing laboratory on the Sprague-Dawley rat over the past three years, the incidence of pup stillbirths in the control groups has typically ranged from 0 to 3.9%. Therefore, this effect is considered to be within normal variations.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

In the 2500 ppm group (both sexes), mean pup body weights were significantly decreased relative to control from lactation Day 7 through weaning, as well as pup body weight change. In the 150 and 500 ppm groups, pup body weights and body weight changes were not affected. The noted effects are considered secondary to general maternal toxicity.

Food consumption and compound intake (if feeding study):

not examined

Description (incidence and severity):

not applicable

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

Description (incidence and severity):

not applicable

Anogenital distance (AGD):

no effects observed

Description (incidence and severity):

There was no statistically significant effect on anogenital distance found.

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):

In the 2500 ppm group organ weights in male and female pups were decreased for the brain, thymus and spleen (absolute weights). For the same animals the relative brain weight was increased, while relative thymus and spleen weights were decreased. Absolute and relative changes in brain weights were consistent with decreases in pup terminal body weights and thus not considered to be directly compound related.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Gross necropsy observations made for pups sacrificed after 21 days and found dead pups were considered to be incidental and not compound-related. However, it should be noted, that lungs that did not float were observed in the stillborn pups described above.

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:

LOAEL

Generation:

F2

Effect level:

2 500 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

organ weights and organ / body weight ratios

Remarks on result:

other: Corresponding to 179.6 mg/kg bw/d (males) and 212.9 mg/kg bw/d (females)

2

Key result

Dose descriptor:

NOAEL

Generation:

F2

Effect level:

500 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other:

Remarks on result:

other: Corresponding to 32.7 mg/kg bw/d (males) and 37.9 mg/kg bw/d (females)

Target system / organ toxicity (F2)

Key result

Critical effects observed:

no

Lowest effective dose / conc.:

2 500 ppm

Organ:

not specified

Overall reproductive toxicity

Key result

Reproductive effects observed:

yes

Lowest effective dose / conc.:

2 500 ppm

Treatment related:

not specified

Relation to other toxic effects:

not specified

Dose response relationship:

not specified

Relevant for humans:

not specified

Applicant's summary and conclusion

Conclusions:

The study was performed under GLP conditions and is in accordance with OECD TG 416 (adopted 1983). No apparent deviations to the current version of the guideline (adopted 2001) were reported. The study is therefore considered reliable and valid. Based on the findings of this study, a NOAEL for parental toxicity of 150 ppm (10 mg/kg bw/day) based on decreased body weight of dams during lactation was established. The NOAEL for effects on neonatal parameters was 150 ppm (10 mg/kg bw/day) based on decreased pup body weight and the subsequent effect on preputial separation. The NOAEL for reproductive effects was 500 ppm (32.7 mg/kg bw/day) based on the increased incidence of stillborn pups, decreased sperm motility and morphology effects at 2500 ppm.