Prometryn

Toxicity to reproduction

Administrative data

Endpoint:

two-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

04 Nov 1987 to 12 Sep 1988

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1990

Report date:

1990

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

EPA OPP 83-4 (Reproduction and Fertility Effects)

Version / remarks:

November 1982

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 418

Version / remarks:

May 12, 1981

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Prometryn

EC Number:

230-711-3

EC Name:

Prometryn

Cas Number:

7287-19-6

Molecular formula:

C10H19N5S

IUPAC Name:

6-(methylsulfanyl)-N2,N4-di(propan-2-yl)-1,3,5-triazine-2,4-diamine

Test material form:

not specified

Test animals

Species:

rat

Strain:

Sprague-Dawley

Remarks:

Crl: CD BR

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Females nulliparous and non-pregnant: yes
- Age at study initiation: (P) males 38 wks and females 37 wks; (F1) 14 wks after weaning
- Weight at study initiation: (P) Males: 171-256 g; Females: 141-201 g; (F1) Males: 139-215 g; Females: 127-177 g
- Housing: rats were caged indivivually (except during the mating, lactation, and weaning periods) in solid bottom cages containing hardwoord chips which were changed weekly
- Diet: Certified Rodent Chow (or its admixture) ad libitum
- Water: tap water ad libitum
- Acclimation period: 1 week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-26
- Humidity (%): 30-70

IN-LIFE DATES:
- P0: From: 4 Nov 1987 To: 29 March 1988
- P1: From: 10 Feb 1988 To: 12 Sep 1988

Administration / exposure

Route of administration:

oral: feed

Vehicle:

acetone

Remarks:

evaporated overnights

Details on exposure:

DIET PREPARATION
Homogeneous blends of the test material in the diet were prepared. The substance was disscoved in acetone and premixed with an appropriate amount of feed. The acetone was evaporatied overnights and the resulting premix was used to prepare the test substance/feed admixturered. The control feed admixture was prepared in a similar manner.

Details on mating procedure:

P0 animals each groups were housed together in a 1:1 (male to female) ratio for mating. Animals were allowed 3 weeks in which to mate and were separated on the day that evidence of mating (sperm and/or copulatory plug) was observed.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Stability studies on admixtures of the test substance in rodent chow over the concentration range of 10 ppm to 50,000 ppm indicated that these mixtures were chemically stable for at least 51 days at room temperature. Analyses of multiple subsamples of test admixtures established acceptable homogeneity. The test substance ranged from 93.2% to 106.4% for these homogeneity analyses. The results of the periodic concentration analyses, performed prior to dosing for admixtures prepared for weeks 1, 5, 9, 11, 17, 18, 25, 33 and 41, showed the concentrations of the test substance in the admixtures to be 93% to 108% of the expected values based on the average results obtained from two or more analyses.

Duration of treatment / exposure:

7 days per week for 2 parental generations of animals and their offspring throughout all phases of the study

Frequency of treatment:

Continuously

Details on study schedule:

The first day of P0 exposure was designated day "0". Following almost 11 weeks of exposure to the appropriate diet. P0 animals each groups were housed together in a 1:1 (male to female) ratio for mating. Animals were allowed 3 weeks in which to mate and were separated on the day that evidence of mating (sperm and/or copulatory plug) was observed. P0 females were allowed to deliver litters.
Following the weaning of the last litter, 30 male and 30 female F1 pups were selected from the available pups in each group to become the second parental generation (P1). Wherever possible, one pup of each sex was randomly selected from each available litter. Litters from which additional pups were selected (in order to achieve 30 per sex per group) were chosen at random. The initiation of the P1 premating phase began with the first recorded body weight. The remaining F1 pups were sacrificed and subjected to a gross internal examination.
One half of the P0 males were sacrificed on day 141 of study and the remaining half were sacrificed on day 142 of study. P0 females were sacrificed after weaning of the F1 pups, half on day 144 of study and half on day 145 of study.
Following their selection, the F1 (P1) animals were allowed 14 weeks of dietary exposure prior to mating. Animals were housed together in the same manner as P0 animals and allowed to deliver their litter. All F2 litters were sacrificed on lactation day 21 and 40 F2 (5/sex/group) were submitted to the Pathology subdivision for necropsy and tissue preservation.
One half of the F1 (P1) males were sacrificed after 145 days on study and the second-half after 146 days on study. One-half of F1 (P1) females were sacrificed after 169 days and the remaining one-half were sacrificed after 172 days.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

10 ppm

Remarks:

Group 2: 0.48-0.93, 0.45-1.09, 0.57-0.88 and 0.54-1.00 mg/kg bw day actual test substance consumption for P0 males, P1 males, P0 females and P1 females, respectively.

Doses / concentrations 2

Dose / conc.:

750 ppm

Remarks:

Group 3: 36.04-70.19, 33.54-80.26, 42.48-64.68 and 43.66-76.04 mg/kg bw day actual test substance consumption for P0 males, P1 males, P0 females and P1 females, respectively.

Doses / concentrations 3

Dose / conc.:

1 500 ppm

Remarks:

Group 4: 72.36-126.76, 68.78-168.88, 87.13-122.78 and 85.87-158.54 mg/kg bw day actual test substance consumption for P0 males, P1 males, P0 females and P1 females, respectively.

No. of animals per sex per dose:

30

Control animals:

yes, plain diet

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS:
All animals were observed daily for changes in appearance and behaviour and twice daily for mortality.

BODY WEIGHT
Body weight data was recorded weekly for the males during the premating period and was recorded weekly thereafter and at terminal sacrifice. Body weight data for the females was recorded weekly during the premating period and on days 0, 7, 14 and 20 of gestation. Female body weights during lactation were recorded on days 0, 7, 14 and 21 for the F0 (P0) generation. During lactation, the F1 (P1) generation female body weights were recorded on days 0, 4, 8, 14 and 21.
P0 females that showed positive evidence of mating but failed to deliver a litter were weighed during the period of presumed gestation and at terminal sacrifice.
F1 females that showed positive evidence of mating but failed to deliver a litter were weighed during the period of presumed gestation until terminal sacrifice.

FOOD CONSUMPTION AND COMPOUND INTAKE
- Feed consumption data was recorded weekly for the males during the premating period. Feed consumption for the females was recorded weekly during the premating period and on days 0, 7, 14 and 20 of gestation.
- Test article consumption was calculated as: Test article consumption = mean daily food consumption / average mean body for interval x dietary concentration

Litter observations:

POSTNATAL OBSERVATIONS OF THE F1 AND F2 GENERATIONS
On the day of delivery, the number and sex of viable pups and the number of stillborn pups were recorded. Newborns were examined for gross abnormalities.
On postnatal day 4, the litters were randomly culled to eight pups (four males and four females), whenever possible.
Pups were observed periodically for changes in appearance and behaviour, and deaths were recorded throughout the lactation period.

IN-LIFE DATA COLLECTION
In- life data for feed consumption, body weight, clinical signs, pup observations, pup sexes and pup weights were collected.
Pup body weights were recorded on lactation days 0, 4, 7, 12, and 21 for the F1 generation and on lactation days 0, 4, 8, 14 and 21 for the F2 generation

Postmortem examinations (parental animals):

All parental animals were sacrificed and necropsied by the Pathology Subdivision. Uterine implantation sites were counted and recorded at necropsy. Any grossly abnormal organ(s) and specimens of vagina, cervix, uterus, ovaries, testes, epididymides, seminal vesicles, prostate, pituitary and coagulating gland were collected from parental animals and fixed in 10% neutral buffered formalin. The fixed tissues from the control and high-dose animals were examined microscopically. These same tissues were examined from 40 randomly selected F2 pups. Grossly abnormal tissues were also examined microscopically. Paired testes and ovaries were weighed.

Postmortem examinations (offspring):

All pups removed from each litter at the time of culling were sacrificed and subjected to a gross internal inspection. This inspection included a brief examination of the abdominal and thoracic cavities, confirmation of sex and a slice through each kidney.
The Pathology Subdivision performed necropsies on 40 (5/sex/group) randomly selected F2 pups.

Statistics:

Statistical analyses were performed as indicated below unless otherwise stated:
- Parental Body Weight, Body Weight Gain, Feed Consumption, Absolute and Relative Organ Weight: One-way Analysis of Variance (ANOVA, Snedecor and Cochran, 1968), Bartlett's Test for Homogeneity of Variance (Snedecor and Cochran, 1968)and Dunnett's Method of Multiple Comparisons between control and treatment groups (Dunnett, 1955, 1964). The calculations derived from the ANOVA were employed in the subsequent Bartlett's and Dunnett's Tests. The primary focus of these analyses is on the results of the Dunnett's comparisons between the control and each of the treated groups. Statistically significant differences between treatment groups (e.g. low dose vs. high-dose group) are not discussed.
- Precoital Interval, Maternal Gestation Duration, Number of Implantations, Viable Fetuses, Stillbirths; Calculated Post- implantation Loss, % Post-implantation Loss, % Stillbirths, Survival Indices, Fertility Indices and Pup Sex Ratios (% males): Chi-squared distributed statistics or Mantel’s trend test
- F1 and F2 generation pup body weights: Healy Analysis (Healy, 1972)

Reproductive indices:

- Post-implantation loss = No. implantations – No. viable pups
- % Post-implantation loss = (No. implantation sites – No. viable pups) / No. implantation sites per dam x 100
- Female fertility = No. pregnant females / No. females with positive evidence for mating x 100
- Female mating index = No. females with positive evidence of mating / No. females cohoused x 100
- Gestation index = No. live litters / No. pregnant females x 100
- Male fertility = No. fertile males / No. males with positive evidence of mating x 100
- Male mating index = No. males with positive evidence of mating / No. males cohoused x 100

Offspring viability indices:

- pre-culling survival = No. pups surviving to day 4 (pre-cull) lactation / No. pups alive day 0 lactation x 100
- post-culling survival = No. pups alive at day 21 lactation / No. pups alive day 4 (post-cull) lactation x 100
- percent males = Total No. viable males (day 0 lactation per group) / Total No. viable pups (day 0 lactation per group) x 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

There were no compound-related clinical observations in this generation, only incidental clinical observations.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

There were no compound-related mortalities in this generation. There were three unscheduled deaths in the P0 generation. One control female (group 1) with a tissue mass (mammary gland adenocarcinoma) was sacrificed in poor physical condition on study day 75 and two 750 ppm (group 3) females were found dead on day 99 and day 102, respectively. The death of one female of group 3, was not associated with any clinical or necropsy observations, while the other female of group 3 was noted as having a dark thick, yellow exudate from its bladder and at necropsy was found to have a bilateral infarction of the kidneys. These deaths were not considered to be treatment-related based on the low incidence of occurrence and a review of the clinical, necropsy, and pathology findings.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

- Males: Mean P0 male body weights in the 10 ppm and 1500 ppm groups were comparable to controls on premating day 0; however a slight, yet statistically significant baseline difference of approximately 3% was observed in the 750 ppm group. This baseline difference persisted throughout the premating period and resulted in statistically lower mean body weights in the 750 ppm group on premating days 7, 21, 28, 35 and 56. These lower mean body weights were generally not associated with reductions in body weight gain or feed consumption and were therefore not related to compound. Compound-related, statistically significant reductions in mean body weight were observed in the 1500 ppm group males on premating days 7 and on all assessment occasion during days 21-70; while significant reductions in mean body weight gain were noted for premating days 0-7, 14-21, 28-35, 49-56 63-70, and overall mean premating body weight gain (premating days 0-70). Non-treatment related, yet statistically significant, fluctuations in mean body weight gain were noted in the 750 ppm group males on premating days 14-21 and 63-70. Group mean body weights were significantly reduced in the 1500 ppm group males during mating and postmating on days 77, 84, 91, 98, 105, 112, 119, 126 and 133; and at terminal necropsy (days 141 or 142). These reductions ranged from 6.3% to 8.2% and were attributed to treatment with the test substance. Although slight, group mean body weight in the 750 ppm group males was also significantly reduced on days 77 and 84. The lower mean weights detected in the 750 ppm group averaged approximately 4% less than controls and were most likely the result of the initial lower mean body weights observed in this group and not a compound effect. The 1500 ppm group mean overall body weight gain (from day 0- termination; was significantly reduced by approximately 12% and mean terminal body weight was significantly reduced by approximately 7.5%. These reductions were attributed to treatment with the test substance No apparent effect on overall mean bodyweight gain or mean terminal body weight was observed at 10 or 750 ppm.
- Females (pre-mating): The 1500 ppm group mean female pre-mating body weights tended to be lower than controls after the first week of premating (2-7%) with a statistically lower mean body weight observed on day 70. In addition the 1500 ppm group mean body weight gain tended to be lower than controls with statistically reduced mean body weight gains detected during premating days 0-7, 42-49 and for mean overall premating weight gain, days 0-70. These reductions in mean body weight parameters occurred concomitantly with treatment-delated reductions in food consumption and were also attributed to treatment. The 10 ppm and 750 ppm group mean body weight and body weight gain were unaffected by treatment. A spurious, yet statistically significant reduction in mean body weight gain was observed at 10 ppm during premating days 63-70. As this decrease was not associated with decreased food consumption or body weight. It was not considered to be toxicologically significant.
- Females (gestation): The 1500 ppm females had consistently lower mean body weights during gestation with treatment-related, statistical decreases observed on gestational days 0, 7, 14 and 20. During gestation mean body weight gain tended to be less than controls at 1500 ppm on days 0-7, 14-20 and 0-20; whereas it was unaffected at ≤ 750 ppm as mean body weight. A spurious, yet statistically significant increase in mean overall gestational body weight gain (days 0-20) was observed at 10 ppm and was not considered to be toxicologically significant.
- Females (lactation): The 1500 ppm animals had significantly reduced mean body weight on lactation day 0 and 14, while significant increases in mean weight gain were observed during lactation days 0-7, 14-21, and 0-21.
- Females (TBW): There were no statistically significant effects on mean terminal body weight in P0 females in this study; although the 1500 ppm group mean terminal body weight was less than the control value by approximately 5%.

See Table 4, 5, 6, 7 and 8 in “Any other information on results incl. tables” for more information.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

- Males: Slight, but statistically significant, treatment-related reductions in mean food consumption of approximately 5-13% were observed in the 1500 ppm group males as compared to controls at the following intervals during the premating period; days 0-7, 21-28, 35-42, 42-49, 49-56 and 63- 70. An isolated, slight, reduction of approximately 5% was observed in the 750 ppm group males during premating days 0-7 and was statistically significant. This reduction was most likely related to initial palatability problems of the dietary admixture upon initial exposure and was not of toxicological significance. No effect on mean food consumption was observed in the 10 ppm group males. The mean of the daily doses of test article ingestion on a mg/kg basis were approximately proportional to dietary concentration increments.
- Females: A statistically significant compound-related reduction in mean food consumption of approximately 9% was observed at 1500 ppm during gestation days 0-7. Mean food consumption was comparable among all groups for the remainder of gestation. No effect on mean food consumption was noted in either the 10 ppm or the 750 ppm group females at any time during the study. The means of daily doses on a mg/kg basis were approximately proportional to dietary concentration increments.

See Table 1, 2 and 3 in “Any other information on results incl. tables” for more information.

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

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

There were no treatment-related histopathological findings in P0 animals.

Histopathological findings: neoplastic:

not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Description (incidence and severity):

There were no significant treatment-related effects on mating as the precoital interval was similar among all groups. The duration of gestation, the reproductive parameters and the mating, fertility, and gestation indices were comparable among the control and substance-treated groups.

Effect levels (P0)

1

Key result

Dose descriptor:

NOAEL

Remarks:

general toxicity

Effect level:

750 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: dietary equivalent to 36.04 - 70.19 mg/kg bw/day for males and 42.48 - 64.68 mg/kg bw for females.

2

Key result

Dose descriptor:

NOAEL

Remarks:

reproductive toxicity

Effect level:

>= 1 500 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reproductive performance

Remarks on result:

other: dietary equivalent to 72.36 - 126.76 mg/kg bw for males and 87.13 - 122.78 mg/kg bw/day for females

Target system / organ toxicity (P0)

Key result

Critical effects observed:

no

Results: P1 (second parental generation)

General toxicity (P1)

Clinical signs:

no effects observed

Description (incidence and severity):

There were no compound-related clinical observations.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

There were no compound-related deaths. Three females did not survive the postmating phase of the study. Two females, one from the 10 ppm group and one from the 750 ppm group were observed in dystocia prior to sacrifice on gestation days 25 and 26 respectively, and another 10 ppm group female was lean cold to touch had a red nasal discharge, paralyzed hindlimbs and a bloody swollen vagina prior to sacrifice on study day 143 and was found to have pyelonephritis of one kidney on histological examination. The first two sacrificed females were attributed to non-treatment related difficulties in labour and delivery. The last sacrificed female was to avoid unnecessary discomfort to the animal caused by non- treatment- related pyelonephritis.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

- Males: Treatment-related reductions in mean body weight were observed in the 750 ppm groups throughout the premating, mating and postmating phases of the study with statistical differences recorded for all assessment occasions during days 28-140 in the 750 ppm group and during days 0- 140 at 1500 ppm and at termination (days 145 or 146) in both groups. Reductions in mean body weight were accompanied by reduced mean body weight gain in both the 750 and 1500 ppm groups during the premating period. These reductions were statistically significant for all assessment intervals during days 0-7, 14-21, 28-35, 0-98 and 0-terminal body weight in the 750 ppm group and during days 0-21, 28-42, 70-77, 0-98, and 0-terminal body weight in the 1500 ppm group. Body weight parameters were unaffected in the 10 ppm group.
- Females: Body weights in the 750 ppm and 1500 ppm groups tended to be lower than controls throughout most of the study. Significant treatment-related reductions in mean body weight were observed in the 750 ppm group during premating (days 49-98)and at terminal necropsy and in the 1500 ppm group during premating (days 21-98) gestation (days 0-20), and lactation (day 0) periods as well as at terminal necropsy. Mean body weight gains were reduced in the 750 ppm and 1500 ppm groups females during the majority of the premating period and were significantly reduced at 750 ppm during premating days 42-49 and overall days 0-98 and at 1500 ppm during premating days 21-35 and 42-49 and overall days 0-98. During gestation body weight gains were comparable among all groups. Lactation body weight gains tended to be greater at 750 ppm and 1500 ppm with mean lactation body weight gain significantly greater during days 0-8, 14-21, as well as mean overall lactation weight gain (days 0- 21) at 1500 ppm; while mean overall lactation body weight gain (day 0-21) was significantly greater at 750 ppm when compared to controls. Mean body weight parameters were unaffected throughout the study at 10 ppm. A single incidental increase in mean body weight gain was observed at 10 ppm during premating days 63-70.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

- Males: Statistically significant treatment-related reductions in mean food consumption were observed on all assessment occasions from premating days 7-14 to 91-98 in the 750 ppm group (6-13%) and premating days 0-7 to 91-98 at 1500 ppm (10-16%). Food consumption at 10 ppm was unaffected by the test substance. The means of daily doses on a mg/kg basis were approximately proportion- al to dietary concentration increments.
- Females (premating): As a result of treatment, mean food consumption in the 750 ppm and 1500 ppm groups tended to be lower than controls throughout the premating period. Statistically significant reductions of 9-13% were observed at 750 ppm over premating days 28-63 and 91-98, while significant reductions of 9-16% were observed at 1500 ppm over premating days 14-21, 28-77 and 84-98. The 10 ppm group female food consumption was unaffected by treatment; however a single, spurious reduction in food consumption (8%) was observed at 10 ppm during days 91-98. In the absence of any noteworthy body weight effects or clinical observations this finding was not considered to be biologically significant. The means of the daily doses on a mg/kg basis were approximately proportional to dietary concentration increments.
- Females (gestation): Mean food consumption during gestation approximated control values on all assessment occasions in the 10 ppm and 750 ppm groups. In the 1500 ppm group mean food consumption was 7-9% less than controls with statistical significance detected for days 0-7. The lower food consumption values at 1500 ppm were attributed to treatment. The means of the daily doses on a mg/kg basis were approximately proportional to dietary concentration increments

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

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

There were no treatment-related effects on mean absolute ovary or testes weight or relative mean ovary weight in the P1 generation; however, in the 1500 ppm group relative mean testes weights were increased as a consequence of reduced mean terminal body weight noted at this dose level. Additionally, the mean absolute testes weight was decreased at 750 ppm but this decrease was attributed to the smaller size of the males in this group and not treatment.

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no treatment-related necropsy observations in the P1 generation parental animals.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

There were no treatment-related histologic findings in the P1 animals.

Histopathological findings: neoplastic:

not examined

Reproductive function / performance (P1)

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

effects observed, non-treatment-related

Description (incidence and severity):

There were no treatment-related effects on mating as the precoital interval was similar for all groups. Male and female mating and fertility indices, the duration of gestation and the gestation index were comparable among the control and treated groups. There were no treatment-related effects on any reproductive parameters examined. However, the mean number of implantation sites and the mean number of viable pups were significantly increased in the 750 ppm and 1500 ppm groups. These increases were incidental to treatment. All other reproductive parameters including the number of stillborn pups, the percentage of stillborn pups, calculated post-implantation loss and percentage post-implantation loss were comparable among all groups.

Effect levels (P1)

1

Key result

Dose descriptor:

NOAEL

Remarks:

general toxicity

Effect level:

10 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: dietary equivalent to 0.45 - 1.09 mg/kg bw/day for males and 0.54 - 1.00 mg/kg bw/day for females

2

Key result

Dose descriptor:

NOAEL

Remarks:

reproductive toxicity

Effect level:

>= 1 500 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reproductive performance

Remarks on result:

other: dietary equivalent to 68.78 - 168.88 mg/kg bw/day for males and 85.87 - 158.54 for females

Target system / organ toxicity (P1)

Key result

Critical effects observed:

no

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Description (incidence and severity):

There were no F1 pup clinical observations in this study.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

no mortality observed

Description (incidence and severity):

There were no treatment related effects on F1 litter size.
Survival indices were comparable among the control and substance-treated groups.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Pup weights were reduced at 1500 ppm for both sexes on all assessment occasions lactation days 0, 4 (pre-cull, post-cull) 7, 14, and 21. Pup weights at 750 ppm also tended to be less than controls during lactation and were significantly reduced on lactation days 14 and 21. The lower pup weights observed through day 7 of lactation in the 750 ppm group were thought to be due to a slightly larger litter size prior to culling. In the 1500 ppm group the lower pup weights were also influenced by a slightly larger litter size and may have been compounded by maternal toxicity. Toward the end of lactation, the lower pup weights were considered due to ingestion of the substance feed admixtures by both the 750 ppm and 1500 ppm pups.

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:

no effects observed

Description (incidence and severity):

There were no treatment related effects on pup sex ratios.

Anogenital distance (AGD):

not examined

Nipple retention in male pups:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no compound- related necropsy findings. Following the selection of the P1 generation, the F1 pups not selected for the second parental generation were sacrificed by subjected to a gross internal inspection. All findings were incidental.

Histopathological findings:

not examined

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Effect levels (F1)

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

750 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

Remarks on result:

other: 33.54 - 80.26 mg/kg bw

Target system / organ toxicity (F1)

Key result

Critical effects observed:

no

Results: F2 generation

General toxicity (F2)

Clinical signs:

no effects observed

Description (incidence and severity):

There were no compound-related clinical signs in pups of the F2 generation.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

no mortality observed

Description (incidence and severity):

There were no treatment-related effects on either parameter; however as a consequence of the incidental increase in the mean number of live foetuses in the 750 ppm and 1500 ppm groups, mean litter size on day 0 was also greater in both groups than the controls. This finding was considered to be incidental.
Survival indices were comparable among the treated and the control groups at both intervals assessed, days 0-4 (pre-cull) or days 4-21 (post-cull) regardless of whether the sexes were analysed separately or as pooled values.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Treatment-related reductions in mean body weight were observed in the 1500 ppm group F2 male and female pups during the majority of the post-natal period [days 4 (pre- and post-cull) 14 and 21]. These lower pup weights were influenced by both a slightly larger litter size and maternal toxicity. The 10 ppm and 750 ppm group F2 pup weights were similar to controls.

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:

no effects observed

Description (incidence and severity):

There were no treatment-related effects on either parameter.

Anogenital distance (AGD):

not examined

Nipple retention in male pups:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

No compound-related necropsy findings were observed. No treatment-related gross alterations were observed in the F2 pups.

Histopathological findings:

no effects observed

Description (incidence and severity):

No treatment-related microscopic alterations were observed in the F2 pups.

Developmental neurotoxicity (F2)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F2)

Developmental immunotoxicity:

not examined

Effect levels (F2)

Key result

Dose descriptor:

NOAEL

Generation:

F2

Effect level:

750 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

Remarks on result:

other: 33.54 - 80.26 mg/kg bw

Target system / organ toxicity (F2)

Key result

Critical effects observed:

no

Overall reproductive toxicity

Key result

Reproductive effects observed:

yes

Lowest effective dose / conc.:

1 500 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:

yes

Any other information on results incl. tables

Table 1. Summary of P0 Male Pre-Mating Food Consumption (grams/day) (Mean ± SD)

Interval	Study days	Dietary level ppm
		Control (0)	10	750	1500
1	0-7	23.65 ± 2.19 (30)$	22.75 ± 1.96 (30)	22.39 ± 1.33* (30)	20.48 ± 1.80* (30)
2	7-14	23.92 ± 1.78 (30)	23.81 ± 1.78 (30)	23.28 ± 1.61 (30)	23.26 ± 2.22 (30)
3	14-21	24.12 ± 2.09 (30)	24.25 ± 1.84 (29)	23.79 ± 1.58 (30)	23.54 ± 2.05 (30)
4	21-28	25.02 ± 2.21 (30)	25.26 ± 1.96 (28)	24.55 ± 1.57 (29)	23.70 ± 2.17* (30)
5	28-35	24.58 ± 2.12 (28)	25.07 ± 2.13  (30)	24.02 ± 1.69 (30)	23.40 + 2.07 (30)
6	35-42	25.44 ± 2.13 (30)	25.91 ± 1.96 (30)	24.49 ± 1.92 (30)	24.01 ± 2.15* (30)
7	42-49	25.56 ± 2.42 (30)	25.66 ± 2.03 (30)	24.74 ± 2.08 (30)	24.07 ± 2.33* (30)
8	49-56	25.84 ± 2.76 (30)	25.69 ± 2.20 (30)	24.56 ± 1.79 (30)	23.79 + 2.46* (30)
9	56-63	25.49 ± 2.73 (30)	25.68 ± 2.33 (30)	24.08 ± 1.94 (30)	24.06 ± 2.26 (30)
10	63-70	26.01 ± 2.28 (29)	25.81 ± 2.55 (30)	24.74 ± 1.83 (30)	24.26 ± 2.46* (30)

$ Numbers in parenthesis ( ) equal number of animals used in mean

* Significantly different from Controls at P ≤ 0.05

 

Table 2. Summary of P0 Female Pre-Mating Food Consumption(grams/day) (Mean ± SD)

Interval	Study days	Dietary level ppm
		Control (0)	10	750	1500
1	0-7	16.05 ± 1.79 (30)$	15.86 ± 1.53 (30)	15.41 ± 1.27 (30)	13.87 ± 1.16* (30)
2	7-14	17.06 ± 2.07 (30)	17.11 ± 2.44 (30)	16.98 ± 1.38 (30)	15.98 ± 1.23 (30)
3	14-21	16.89 ± 2.15 (30)	16.72 ± 1.97 (30)	17.06 ± 1.49 (30)	16.16 ± 2.74 (28)
4	21-28	17.32 ± 2.16 (30)	17.35 ± 2.13 (30)	17.51 ± 1.84 (30)	16.23 ± 1.42 (30)
5	28-35	16.91 ± 2.10 (30)	17.30 ± 2.13 (30)	17.41 ± 1.84 (29)	15.51 ± 1.05* (29)
6	35-42	17.19 ± 1.76 (30)	17.14 ± 2.36 (30)	17.63 ± 2.16 (30)	15.97 ± 1.35* (30)
7	42-49	17.42 ± 2.38  (30)	17.96 ± 2.60 (30)	17.87 ± 2.19 (30)	15.97 ± 2.12* (30)
8	49-56	17.94 ± 2.56 (30)	17.26 ± 2.18 (30)	17.07 ± 2.49 (30)	15.59 ± 1.12* (30)
9	56-63	17.55 ± 2.50 (30)	16.86 ± 2.26 (30)	16.59 ± 1.70 (30)	15.40 ± 1.22* (30)
10	63-70	17.61 ± 2.32 (30)	16.93 ± 1.96 (30)	17.05 ± 1.94 (30)	15.89 ± 1.13* (30)

$ Numbers in parenthesis ( ) equal number of animals used in mean

* Significantly different from Controls at P ≤ 0.05

Table 3. Summary of P0 Female Gestation Food Consumption(grams/day) (Mean ± SD)

Interval	Days of Gestation	Dietary level ppm
		Control (0)	10	750	1500
1	0-7	22.22 ± 2.23 (29)$	21.19 ± 1.96 (24)	20.62 ± 2.21 (29)	19.23 ± 2.21* (29)
2	7-14	21.61 ± 2.80 (29)	21.55 ± 2.87 (24)	21.86 ± 1.92 (29)	20.77 ± 1.41 (29)
3	14-20	22.87 ± 2.83 (29)	23.42 ± 1.97 (24)	22.45 ± 3.49 (28)	21.53 ± 3.20 (29)

$ Numbers in parenthesis ( ) equal number of animals used in mean

* Significantly different from Controls at P ≤ 0.05

 

Table 4. Summary of P0 Male Pre-Mating Body Weights (grams) (Mean ± SD)

Study days	Dietary level ppm
	Control (0)	10	750	1500
0	220.67 ± 11.27 (30)$	218.57 ± 9.03 (30)	213.80 ± 12.71* (30)	221.27 ± 10.94 (30)
7	273.67 ± 15.10 (30)	271.03 ± 13.89 (30)	265.10 ± 10.96* (30)	262.83 ± 13.38* (30)
14	319.13 ± 16.81 (30)	320.70 ± 14.78 (30)	311.37 ± 12.60 (30)	310.03 ± 19.30 (30)
21	364.47 ± 20.62 (30)	364.93 ± 19.08 (30)	351.80 ± 13.74* (30)	348.23 ± 22.23* (30)
28	399.57 ± 24.70 (30)	399.03 ± 22.01 (30)	383.50 ± 16.61* (30)	380.50 ± 26.00* (30)
35	432.97 ± 28.80 (30)	434.10 ± 25.73 (30)	415.70 ± 17.76* (30)	410.20 ± 28.85* (30)
42	455.80 ± 29.91 (30)	461.00 ± 27.45 (30)	441.13 ± 20.38 (30)	432.40 ± 30.92* (30)
49	481.57 ± 34.51 (30)	486.27 ± 30.25 (30)	464.50 ± 22.36 (30)	456.77 ± 34.62* (30)
56	507.40 ± 38.10 (30)	509.43 ± 30.84 (30)	486.73 ± 25.76* (30)	477.63 ± 37.22* (30)
63	522.40 ± 40.78 (30)	528.83 ± 34.29 (30)	504.53 ± 27.53 (30)	495.13 ± 38.69* (30)
70	546.90 ± 42.46 (30)	549.47 ± 39.14 (30)	524.63 ± 29.06 (30)	509.77 ± 41.21* (30)

$ Numbers in parenthesis ( ) equal number of animals used in mean

* Significantly different from Controls at P ≤ 0.05

 

Table 5. Summary of P0 Male Mating Phase (77-98) and Post-Mating Phase (105-141,142) Body Weights(grams) (Mean ± SD)

Study days	Dietary level ppm
	Control (0)	10	750	1500
77	545.73 ± 44.19 (30)$	547.50 ± 38.86 (30)	521.57 ± 30.25* (30)	511.60 ± 42.24* (30)
84	558.70 ± 46.35 (30)	559.07 ± 40.86 (30)	533.50 ± 29.38* (30)	522.57 ± 43.81* (30)
91	579.87 ± 49.25 (30)	578.87 ± 45.55 (30)	553.63 ± 32.41 (30)	539.73 ± 46.92* (30)
98	590.23 ± 51.84 (30)	592.33 ± 47.41 (30)	566.83 ± 34.14 (30)	551.23 ± 49.44* (30)
105	602.13 ± 53.35 (30)	605.87 ± 45.29 (30)	578.90 ± 34.90 (30)	561.10 ± 49.39* (30)
112	613.23 ± 56.41 (30)	616.73 ± 47.69 (30)	585.70 ± 37.43 (30)	569.30 ± 50.76* (30)
119	618.90 ± 57.37 (30)	622.23 ± 48.89 (30)	592.50 ± 38.41 (30)	572.47 ± 52.20* (30)
126	635.90 ± 56.05 (30)	637.40 ± 48.05 (30)	606.03 ± 37.47 (30)	583.87 ± 52.06* (30)
133	646.87 ± 60.95 (30)	643.97 + 52.01 (30)	615.00 ± 40.78 (30)	596.63 ± 55.88* (30)
TBW ^	631.27 ± 60.92 (30)	632.27 ± 52.45 (30)	601.57 ± 41.04 (30)	583.73 ± 55.13* (30)

$ Numbers in parenthesis ( ) equal number of animals used in mean

^ TBW = Terminal body weight – Day 141 or 142 of study

* Significantly different from Controls at P ≤ 0.05

 

Table 6. Summary of P0 Female Pre-Mating Body Weights (grams) (Mean ± SD)

Study days	Dietary level ppm
	Control (0)	10	750	1500
0	167.37 ± 14.63 (30)$	169.07 ± 11.52 (30)	169.97 ± 11.47 (30)	170.90 ± 9.32 (30)
7	189.60 ± 17.18 (30)	190.37 ± 14.89 (30)	187.83 ± 13.35 (30)	184.23 ± 10.62 (30)
14	212.63 ± 21.27 (30)	211.53 ± 17.51 (30)	213.13 ± 15.83 (30)	206.40 ± 12.19 (30)
21	225.40 ± 23.45 (30)	227.27 ± 17.88 (30)	228.73 ± 16.27 (30)	220.23 ± 11.88 (30)
28	238.97 ± 26.81 (30)	241.07 ± 22.10 (30)	241.20 ± 18.84 (30)	229.77 ± 15.05 (30)
35	249.00 ± 29.07 (30)	253.77 ± 24.34 (30)	254.50 ± 22.58 (30)	243.10 ± 15.03 (30)
42	259.30 ± 29.45 (30)	263.27 ± 26.79 (30)	264.80 ± 25.04 (30)	250.03 ± 16.57 (30)
49	269.20 ± 32.53 (30)	276.20 ± 29.09 (30)	275.17± 27.30 (30)	254.73 ± 17.78 (30)
56	278.67 ± 37.06 (30)	283.97 ± 30.39 (30)	282.83 ± 29.90 (30)	261.87 ± 16.60 (30)
63	288.60 + 39.57 (30)	293.43 ± 34.19 (30)	290.47 ± 30.56 (30)	269.90 ± 19.96 (30)
70	298.63 ± 41.69 (30)	299.27 ± 33.96 (30)	298.87 ± 33.64 (30)	278.03 ± 20.99* (30)

$ Numbers in parenthesis ( ) equal number of animals used in mean

* Significantly different from Controls at P ≤ 0.05

 

Table 7. Summary of P0 Female Gestation Body Weights (grams) (Mean ± SD)

Gestation Day	Dietary level ppm
	Control (0)	10	750	1500
0	307.90 ± 41.66 (29)$	306.29 ± 35.44 (24)	301.59 ± 25.50 (29)	283.31 ± 20.26* (29)
7	335.03 ± 44.18 (29)	339.71 ± 31.23 (24)	328.76 ± 27.87 (29)	308.10 ± 21.93* (29)
14	362.52 ± 46.27 (29)	370.92 ± 32.83 (24)	359.10 ± 28.55 (29)	336.83 ± 21.00* (29)
20	431.69 ± 52.65 (29)	446.54 ± 30.84 (24)	427.14 ± 39.66 (28)	400.62 ± 30.23* (29)

$ Numbers in parenthesis ( ) equal number of animals used in mean

* Significantly different from Controls at P ≤ 0.05

 

Table 8. Summary of P0 Female Lactation Body Weights (grams) (Mean ± SD)

Gestation Day	Dietary level ppm
	Control (0)	10	750	1500
0	339.34 ± 45.87 (29)$	347.04 ± 35.32 (26)	333.11 ± 29.88 (28)	308.34 ± 23.15* (29)
7	345.52 ± 40.34 (29)	354.38 ± 27.15 (26)	346.26 ± 25.92 (27)	328.90 + 20.94 (29)
14	358.76 ± 35.16 (29)	370.15 ± 29.11 (26)	358.78 + 23.74 (27)	336.24 ± 23.50* (29)
20	342.83 ± 31.92 (29)	349.65 ± 28.28 (26)	350.22 ± 20.85 (27)	340.72 ± 17.8 (29)

$ Numbers in parenthesis ( ) equal number of animals used in mean

* Significantly different from Controls at P ≤ 0.05

Table 9. Summary of F0 (P1) generation precoital interval.

Dietary level (ppm)	Percent of females mating during cohabitation days
	1 – 4	5 – 8	9 – 12	13 – 16	17 – 21
0	82.8 (24/29)a, b	6.9 (2/29)	3.4 (1/29)	0 (0/29)	6.9 (2/29)
10	90 (27/30)c	3.3 (1/30)	0 (0/30)	3.3 (1/30)	3.3 (1/30)
750	93.3 (28/30)	6.7 (2/30)	0 (0/30)	0 (0/30)	0 (0/30)
1500	96.7 (29/30)	0 (0/30)	0 (0/30)	3.3 (1/30)	0 (0/30)

a) Numbers in oarentheses ( ) represents the number of females with evidence of mating during that interval over the total number of females in that group showing evidence of mating during the entire mating period

b) 0 ppm – one animal sacrificed after 1 day of mating

c) 10 ppm – no sperm was observed in two females. The mating date was estimated by subtracting 23 days from the date of pup delivery

 

Table 10. Summary of F0 generation reproductive parameters (mean and standard deviation)(a)

	Dietary level (ppm)
Parameter	Control (0)	10	750	1500
Number of pregnant females	29	26	29	29
Number of viable litters	29	26	28	29
Number of implantation sites	15.97 ± 2.26	15.92 ± 2.37	16.66 ± 2.24	16.66 ± 1.76
Number of stillbirths	0.31 ± 0.71	0.31 ± 0.79	0.11 ± 0.31	0.38 ± 1.01
% stillbirths	2.21 ± 4.74	2.06 ± 5.34	0.68 ± 2.01	2.43 ± 6.41
Number of viable newborns	14.21 ± 2.11	14.81 ± 2.81	15.25 ± 2.17	15.24 ± 2.03
Post-implantation loss (b)	1.76 ± 1.33	1.15 ± 1.08	1.61 ± 1.37	1.45 ± 1.66
% post-implantation loss (b)	10.85 ± 7.83	7.74 ± 8.25	9.42 ± 8.41	8.44 ± 9.42

a) Although means and standard deviations are reported, the actual statistical analysis is based on non-parametric methods

b) When the number of viable pups exceeds the number of implants, the post-implantation loss was calculated as zero to ensure meaningful biological interpretation of statistical analysis

 

Table 11. Summary of the F0 Generation Reproductive and Fertility Indices (c)

Parameter	Dietary level (ppm)
	Control (0)	10	750	1500
Female fertility	29/29 (a) (100)	26/30 (b) (86.7)	29/30 (96.7)	29/30 (96.7)
Female mating index	29/29 (a) (100)	30/30 (b) (100)	30/30 (100)	30/30 (100)
Gestation index	29/29 (a) (100)	26/26 (100)	28/29 (96.6)	29/29 (100)
Male fertility	29/29 (a) (100)	26/30 (b) (86.7)	29/30 (96.7)	29/30 (96.7)
Male mating index	29/29 (a) (100)	30/30 (b) (100)	30/30 (100)	30/30 (100)

a) One female sacrificed on study day 75 after 1 day of mating.

b) Two females were not observed as sperm positive but delivered litters

c) Data expressed as group ratios and percentages (in parenthesis)

 

Table 12. Summary of the F1 Litter size (Day 0 lactation) survival indices and sex ratios

Parameter	Dietary level (ppm)
	Control (0)	10	750	1500
Number of litters born	29	26	28	29
Mean litter size (excluding stillbirths)	14.21	14.81	15.25	15.24
Mean number of stillbirths	0.31	0.31	0.11	0.38
Number of viable males (Day 0)	223	183	213	230
Number of viable females (Day 0)	189	202	214	212
Survival indices Sexes pooled
Mean % pups surviving, days 0 – 4 (pre-cull)	99.35	94.64	95.13	97.31
Mean % pups surviving, days 4 – 21 (post-cull)	99.14	99.04	99.34	99.55
Survival indices by sex
Mean % males surviving, Days 0 – 4 (pre-cull)	98.99	96.05	95.39	97.51
Mean % males surviving, Days 4 - 21 (post-cull)	99.14	99.04	99.07	99.11
Mean % females surviving, Days 0 – 4 (pre-cull)	99.43	93.83	95.11	97.21
Mean % females surviving, Days 4 - 21 (post-cull)	99.14	99.04	100	100
Sex ratio Day 0 Lactation	54.13	47.53	49.88	52.04

 

Table 13. Summary of F1 male pup body weights (grams, (a) mean and standard error)

Postnatal days	Dietary levels (ppm)
	Control (0)	10	750	1500
0	6.34 ± 0.09 (28)(b)	6.38 ± 0.10 (26)	6.18 ± 0.09 (28)	6.05 ± 0.09 *
4 (pre-culling)	9.53 ± 0.21 (29)	9.57 ± 0.23 (26)	9.02 ± 0.22 (27)	8.9 ± 0.21 *
4 (post-culling)	9.45 ± 0.22 (29)	9.7 ± 0.23 (26)	8.99 ± 0.22 (27)	8.85 ± 0.22 (29)
7	15.73 ± 0.36 (29)	15.99 ± 0.38 (26)	14.94 ± 0.37	14.62 ± 0.36 *
14	33.35 ± 0.56 (29)	33.56 ± 0.59 (26)	31.66 ± 0.58 *	30.22 ± 0.56 *
21	55.03 ±1 (29)	55.96 ± 1.06 (26)	51.44 ± 1.04 * (27)	48.46 ± 1.02 * (29)

a) Values for the weighted means and standard errors are derived from the Healy analysis

b) Numbers in parenthesis ( ) equal number of litters used in mean

* Statistically significant at P ≤ 0.05.

 

Table 14. Summary of F1 female pup body weights (grams, (a), mean and standard error)

Postnatal days	Dietary levels (ppm)
	Control (0)	10	750	1500
0	5.98 ± 0.09 (28) (b)	6.09 ± 0.1 (26)	5.83 ± 0.09 (28)	5.68 ± 0.09 * (28)
4 (pre-culling)	9.13 ± 0.2 (29)	9.25 ± 0.21 (26)	8.66 ± 0.21 (27)	8.44 ± 0.2 * (29)
4 (post-culling)	9.14 ± 0.2 (29)	9.32 ± 0.22 (26)	8.72 ± 0.21 (27)	8.44 ± 0.2 * (29)
7	15.26 ± 0.34 (29)	15.18 ± 0.36 (26)	14.43 ± 0.35 (27)	13.87 ± 0.34 * (29)
14	32.25 ± 0.56 (29)	32.17 ± 0.59 (26)	30.58 ± 0.58 * (27)	29.04 ± 0.56 * (29)
21	52.91 ± 0.94 (29)	53.27 ± 1 (26)	49.32 ± 0.97 * (27)	47.02 ± 0.94 (29)

a) Values for the weighted means and standard errors are derived from the Healy analysis

b) Numbers in parenthesis ( ) equal number of litters used in mean

* Statistically significant at P ≤ 0.05.

 

Table 15. Summary of mean F1 litter size and weight (a)

Dietary level	0		4 (PR) (b)		4 (P0) (c)		7		14		21
	Size	Weight	Size	Weight	Size	Weight	Size	Weight	Size	Weight	Size	Weight
0	14.21	6.18	14.10	9.34	8	9.3	7.93	15.50	7.93	32.81	7.93	53.98
10	14.81	6.25	14	9.44	7.81	9.52	7.73	15.60	7.73	32.87	7.73	54.67
750	15.25	6.01	15.26	8.86	8	8.86	7.96	14.68	7.96	31.11	7.96	50.36
1500	15.18	5.88	14.79	8.69	8	8.64	8	14.24	7.97	29.62	7.83	47.9

a) Size – mean number of viable pups per viable litter; weight – mean pup weight (g) per viable litter

b) PR – pre-culling

c) P0 – post-culling

 

Table 16. Summary of F1 Female Gestation Food Consumption (g/day), mean and SD.

Interval	Study days	Dietary levels (ppm)
		0	10	750	1500
1	0 – 7	21.94 ± 2.67 (21)	21.40 ± 1.91 (23)	20.37 ± 2.84 (28)	20.04 ± 1.58 * (24)
2	7 – 14	23.05 ± 2.68 (21)	23.36 ± 2.25 (23)	22.56 ± 2.83 (28)	21.47 ± 1.46 (24)
3	14 – 20	25.26 ± 2.77 (21)	25.73 ± 2.37 (23)	24.86 ± 3.47 (28)	23.45 ± 1.67 (24)

Number in parenthesis ( ) equal number of animals used in mean.

* Significantly different from Controls at P ≤ 0.05

 

Table 17. Summary of F1 (P2) generation precoital interval.

Dietary level (ppm)	Percent of females mating during cohabitation days
	1-4	5-8	9-12	13-16	17-21
0	92 (23/25^b)^a	0	0	4 (1/25)	4 (1/25)
10	82.1 (23/28)	3.6 (1/28)	0	3.6 (1/28)	10.71 (3/28)
750	86.7 (26/30)	0	0	10 (3/30)	3.3 (1/30)
1500	93.3 (28/30)	3.3 (1/30)	0	0	3.3 (1/30)

(a) Number in parenthesis represents the number of females with evidence of mating during that interval over the total number of females in that group showing evidence of mating during the entire mating period.

(b) In one female sperm was not observed in the vaginal washings; however she delivered a viable litter

 

Table 18. Summary of F1 generation reproductive parameters (mean ± standard derivation)

Parameter	Dietary level (ppm)
	Control (0)	10	750	1500
Number of pregnant females	22	23	28	24
Number of viable litters	21	20	27	24
Number of implantation sites	14.4 ± 4.57	11.83 ± 5.7	15.50 ± 3.54*	16.75 ± 3.98*
Number of stillbirths	0.18 ± 0.5	0.23 ± 0.53	0.37 ± 1.28	0.21 ± 0.41
% stillbirths	1.64 ± 4.38	1.74 ± 3.99	2.07 ± 6.76	1.33 ± 2.75
Number of viable newborns	12.05 ± 4.98	10.82 ± 5.68	14.22 ± 3.48*	14.83 ± 3.58*
Post-implantation loss (a)	2.19 ± 2.6 (b)	1 ± 1.2	1.48 ± 1.81	1.92 ± 2.8
% Post-implantation loss	18.96 ± 24.90	15.97 ± 28.47	9.09 ± 10.68	10.83 ± 13.08

(a) When the number of viable pups and stillbirths exceeded the number of implants, the post-implantation loss was calculated as zero to ensure meaningful biological interpretation of the statistical analysis.

* Statistically different at P ≤ 0.05

 

Table 19. Summary of F1 generation reproductive and fertility indices.

Parameter	Dietary level – ppm
	Control (0)	10	750	1500
Female fertility	22/25 (88)	23/28 (82.1)	28/30 (93.3)	24/30 (80)
Female mating index	25/30 (83.3)	28/30 (93.3)	30/30 (100)	30/30 (100)
Gestation index	21/22 (95.5)	20/23 (87)	27/28 (96.4)	24/24 (100)
Male fertility	22/25 (88)	23/28 (82.1)	28/30 (93.3)	24/30 (80)
Male mating index	25/30 (83.3)	28/30 (93.3)	30/30 (100)	30/30 (100)

 

Table 20. Summary of F2 litter size (Day 0 lactation). Survival indices and sex ratios.

Parameter	Dietary level (ppm)
	Control (0)	10	750	1500
Number of litters born	21	20	27	24
Mean litter size (day 0 lactation; excluding stillbirths)	12.62	11.9*	14.22*	14.83*
Mean no. stillbirths	0.18	0.23	0.37	0.21
Number of viable males (day 0)	126	112	195	167
Number of viable females (day 0)	139	126	189	189
Survival indices sexes pooled:
Mean % pups surviving, Days 0-4 (pre-cull)	99.68	93.66	96.86	96.55
Mean % pups surviving, Days 4-21 (post-cull)	99.38	97.37	100	98.96
Survival Indices by sex:
Mean % males surviving, Day 0-4 (pre-cull)	100	93.11	96.08	96.47
Mean % males surviving, Days 4-21 (post-cull)	98.68	98.68	100	100
Mean % females surviving, Day 0-4 (pre-cull)	99.38	94.55	97.60	96.05
Mean % females surviving, Days 4-21 (post-cull)	100	96.05	100	98.26
Sex ratio Day 0 Lactation (% males)	47.55	47.06	50.78	46.91

 

Table 21. Summary of mean F2 litter size and weight (day of lactation).

Dietary level (ppm)	0		4 (PR)		4 (PO)		7		14		21
	Size	Weight	Size	Weight	Size	Weight	Size	Weight	Size	Weight	Size	Weight
0	12.62	6.4	12.57	9.5	7.33	9.47	7.3	16.68	7.29	30.44	7.4	48.96
10	11.9	6.48	12.05	9.13	7.37	9.17	7.26	16.34	7.16	30.37	7.16	49.51
750	14.22	6.04	13.7	8.92	7.85	8.95	7.85	16.27	7.85	29.26	7.85	46.96
1500	14.83	5.97	14.17	8.48	7.83	8.44	7.79	15.09	7.79	27.26	7.75	43.81

Applicant's summary and conclusion

Conclusions:

The lowest NOAEL for parental, systemic toxicity was considered to be 10 ppm for the P1 generation males (0.48 - 0.93 mg/kg bw/day) and 750 ppm for the P1 generation females (43.66 - 76.04 mg/kg bw/day).
The NOAEL for reproduction toxicity was considered to be in excess of 1500 ppm for P0 and P1 generation males and females (corresponding to average doses of 99.6 mg/kg bw/day in males and 105.0 mg/kg bw/day in females).
The NOAEL for foetal toxicity was considered to be 750 ppm for F1 and F2 generations (corresponding to average doses of 53.1 mg/kg bw/day in males and 53.6 mg/kg bw/day in females).

Executive summary:

In this two-generation reproductive toxicity study (GLP and EPA 83-4) the test substance was administered in the diet to three groups of male and female rats at concentrations of 10, 750, or 1500 ppm for two successive generations (P0, P1). A fourth group of rats received diet without the test substance for two generations and served as controls. There were no compound-related mortalities or clinical observations during treatment of either parental (P0 or P1) generation. Reductions in mean body weight parameters were observed in the 1500 ppm P0 and the 750 ppm and 1500 ppm P1 males and females during the premating period and frequently persisted into gestation and lactation for females. Treatment-related reductions in food consumption were noted at 1500 ppm of the P0 generation and at 750 ppm and 1500 ppm in the P1 generation for both sexes during premating. P0 and P1 female food consumption during gestation was also reduced at 1500 ppm. Mating and fertility indices were comparable for the control and treated groups of both generations. There were no treatment-related effects on the duration of gestation, fertility indices, or any of the reproductive parameters. Mean ovary and testes weights were comparable among treated and control groups. The mean relative testes weight was significantly increased at the 1500 ppm dose as a result of the reduced mean terminal body weight in this group. Necropsy and histopathological examinations of the P0 and P1 parental generation animals and selected F2 pups did not reveal any effects attributable to treatment. Litter size, sex ratio, and postnatal survival indices of the F1 and F2 generation were unaffected by treatment. There were no treatment-related clinical observations, necropsy observations, or malformations. The 750 ppm and 1500 ppm F1 and the 1500 ppm F2 pup weights were reduced at most intervals during lactation. The lower pup weights were influenced by a slightly larger litter size, maternal toxicity, and ingestion of the test substance by the pups. Pups were similar to controls at 10 ppm.
The study data indicate that the test substance did not cause any adverse effects on the overall reproductive performance in rats fed concentrations up to and including 1500 ppm for two generations. Under the conditions of this study, the no observable effect level (NOEL) was 10 ppm.

The NOAEL for systemic toxicity was considered to be 750 ppm for P0 males and females (on average 53.1 and 53.6 mg/kg bw/day, respectively) and 10 ppm for the P1 generation males (on average 0.68 mg/kg bw/day) and 750 ppm for the P1 generation females (on average 59.9 mg/kg bw/day). The NOAEL for reproduction toxicity was considered to be in excess of 1500 ppm for P0 and P1 generation males and females (99.6 mg/kg bw/day in males and 105.0 mg/kg bw/day in males and females, respectively). The NOAEL for feotal toxicity was considered to be 750 ppm for F1 and F2 generations (53.1 mg/kg bw/day in males and 53.6 mg/kg bw/day in females, respectively).