2-Pyridinesulfonamide, N-[[(4,6-dimethoxy-2-pyrimidinyl)amino]carbonyl]-3-(2,2,2-trifluoroethoxy)-, sodium salt (1:1)

Toxicity to reproduction

Administrative data

Endpoint:

two-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

11 August 1997 to 21 January 1999

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study was performed to GLP and in line with standardised guidelines OECD 416, EPA OPP 83-4 and EPA OPPTS 870.3800 with no deviations thought to impact on the reliability of the presented results.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2000

Report date:

2000

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPP 83-4 (Reproduction and Fertility Effects)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3800 (Reproduction and Fertility Effects)

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: J-MAFF 59 NohSan No. 4200

Deviations:

no

Test guideline 5

Qualifier:

according to guideline

Guideline:

EU Method B.35 (Two-Generation Reproduction Toxicity Test)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

sodium 4,6-dimethoxy-N-({[3-(2,2,2-trifluoroethoxy)pyridin-2-yl]sulfonyl}carbamoyl)pyrimidin-2-aminide

EC Number:

688-332-8

Cas Number:

199119-58-9

Molecular formula:

C14H13F3N5O6SNa

IUPAC Name:

sodium 4,6-dimethoxy-N-({[3-(2,2,2-trifluoroethoxy)pyridin-2-yl]sulfonyl}carbamoyl)pyrimidin-2-aminide

Test material form:

solid: particulate/powder

Details on test material:

- Physical state: solid (powder)
- Storage condition of test material: room temperature

Test animals

Species:

rat

Strain:

other: Tif:RAIf

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: 6-7 weeks
- Housing: individually
- Diet: pelleted, certified standard diet available ad libitum
- Water: tap water ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 50 ± 20 %
- Air changes (per hr): about 16 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours dark / 12 hours light

IN-LIFE DATES: From11 August 1997 to 12 May 1998

Administration / exposure

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: Ground feed pellets were mixed with the appropriate quantity of test material, hydrated and repressed to pelets.

DIET PREPARATION
- Rate of preparation of diet (frequency): approximately monthly
- Storage temperature of food: room temperature (as unopened sacks or in stainless steel bins with dust-tight lids)

Details on mating procedure:

- M/F ratio per cage: 1/1
- Housing: each mating pair was housed in a cage separated by an aluminium divider containing a closed guillotine door. Pairs were cohabited one day before mating until midnight when restricted mating access was allowed by activating the guillotine door. The guillotine door was programmed to automatically open at midnight allowing access between the males and female sections of the cage. Beginning at 06.30 the the female was assessed for proof of pregnancy. Where no proof of mating was apparent, males and females were separated behind the guillotine door and the process was repeated. If no evidence of mating occurred the stage of estrous cycle was obtained.
- Length of cohabitation: up to 21 days
- Proof of pregnancy: vaginal plug or sperm in vaginal smear
- After successful mating each pregnant female was caged: individually

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The stability of the test material was determined in a previous rangefinding test; this also helped inform on the dose concentrations employed in this study.

For analytical verification of doses, the following samples were taken in duplicate from the first feed batch: control diet (500 g powder and 500 g pellets); treated diet - ground feed mix (200 g per dose prior to pressing); treated diet - pellets (3 x 200 g per dose - samples from beginning, middle and end of pelleting). In addition, 200 g treated pellets (each dose) and 500 g control pellets were samples at the middle of all other pressing runs.

- Analytical Method:
10 g of diet were extracted with 0.2 % phosphoric acid in water and acetonitrile. Aliquots of the extracts were appropriately diluted with a mixture of 0.2 % phosphoric acid in water for HPLC + acetonitrile for HPLC 60 vol. + 40 vol. and the test material was determined by HPLC using UV detection.

Duration of treatment / exposure:

Two generations (a total of 39 weeks)

Frequency of treatment:

Daily

Details on study schedule:

- F1 parental animals not mated until 10 weeks after selected from the F1 litters.

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 500, 1000, 8000 and 12000 ppm
Basis:
nominal in diet

No. of animals per sex per dose:

30 males and 30 females (P and F1)

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: Doses were based on findings of a rangefinding rat reproduction study and in a 3 month dietary toxicity study in rats. In the rangefinding study, test material was administered at dietary dose levels of 0, 1000, 8000 and 16000 ppm. Bodyweight gains were impaired in high dose group animals and pups relative to controls. At 8000 ppm, bodyweight gain deficits were seen in females only during lactation and in pups during week 3 postpartum. Food consumption was also reduced in high dose group animals but no other treatment-related effects were noted during the study. In the oral toxicity study, test material was administered as dietary dose levels of 0, 100, 1000, 4000, 8000 and 16000 ppm for 13 weeks. No effects on bodyweight gain were noted in any other experimental group. The bodyweight gain data, together with a number of other treatment related effects led to the conclusion that the maximum tolerated dose was met of even slightly exceeded at 16000 ppm. At 8000 ppm no treatment related effects were observed.
In consideration of the findings, the following dose levels were selected for the two-generation study:
500 ppm - this dose was expected to cause no observable effects
1000 ppm - this dose was expected to cause no or minimal observable effects
8000 ppm - this dose was expected to cause minimal observable effects
16000 ppm - this dose was expected to cause observable effects, but no or few fatalities

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily, at least 6 hours apart

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- Time schedule: weekly (except during cohabitation for mating) based on the food weighed in and the food weight out.
- Mean daily food consumption: calculated by dividing the food consumption per interval by the number of days in the interval.
- Mean daily test material intake: calculated by multiplying the mean daily food consumption for an interval by the nominal test material concentration, then dividing it by bodyweight at the beginning of the interval.

Oestrous cyclicity (parental animals):

The length and succession of cycle stages was evaluated for all females during the three weeks preceding the mating period and throughout cohabitation until evidence for positive mating was found. The beginning of a cycle was defined as the first day estrus was coded. The end of the cycle was defined as the day before the next day of estrus. The length of the cycle was defined as the number of days from the beginning of the cycle to the end of the cycle, inclusive.

Sperm parameters (parental animals):

Parameters examined in all male parental generations: sperm count in testes, sperm count in epididymides, sperm motility and sperm morphology.

Litter observations:

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

PARAMETERS EXAMINED
The following parameters were examined in F1 / F2 offspring: number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities, righting reflex and eye opening.

PUP SEXUAL MATURATION ASSESSMENT:
Each F1 pup selected for mating was examined as follows and the day and bodyweight when the criteria were reached were recorded:
- Preputial separation (males): was evaluated daily beginning day 24 postpartum; the criterion was prepuce remains open after gentle pressure
- Vaginal opening (females): was evaluated once daily beginning day 30 postpartum; the criterion was vagina remains open after gentle pressure

GROSS EXAMINATION OF DEAD PUPS:
Yes

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals (at time of weaning of F1 generation).
- Maternal animals: All surviving animals (at time of premating of F1 generation).

GROSS NECROPSY
- Gross necropsy was performed.

HISTOPATHOLOGY / ORGAN WEIGHTS
- The following tissues were removed and weighed: adrenals, brain, kidneys, liver, spleen, thymus, ovaries, uterus, cervix, oviducts, lesions, pituitary, testes, epididymides, seminal vesicles, prostate and coagulating gland.
- The following tissues were examined for pathology: adrenals, liver, vagina, ovaries, uterus, cervix, oviducts, lesions, pituitary, testes, epididymides, seminal vesicles, prostate and coagulating gland. Any other macroscopic abnormality was examined for pathology.
Full histopathological examination was performed on the organs/tissues indicated above in all control and 12000 ppm F0 and F1 adult animals.

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring not selected as parental animals of for special evaluation were sacrificed after the last litter was weaned (day 24-34 postpartum); all F2 offspring not selected for special evaluation were sacrificed on lactation day 22. A complete gross necropsy was conducted on all live, stillborn and sacrificed pups.
- These animals were subjected to postmortem examinations consisting of examination of the main organs of the thoracic and abdominal cavities, with special attention directed to the organs of the reproductive system.

PUP TISSUE SAMPLING AND ORGAN WEIGHTS
- at day 22 postpartum, if a F1 litter had at least 2 male or female pups, one pup per sex was randomly selected for special evaluation. For F2 litters, if there was only one male or female pup, this pup was selected, and otherwise the selection was random. These pups were weighed and necropsied. The following organs were preserved: liver, testes, macroscopic abnormalities, and in the case of the brain, spleen and thymus, were weighed as well.

Statistics:

Statistical analysis of continuous data was performed using the Analysis of Variance Procedure followed by the Dunnett t-Test in case of a significant result. Categorical data were analysed using a chi-square test followed by the Fisher Exact test with the Bonferroni correction in case of a significant result in he chi-square test.

Reproductive indices:

- Female mating: percent of females with sign of positive mating (or pregnant without signs of mating) out of total females cohabited.

- Female fertility (fecundity): percent of females with confirmed pregnancy out of total females with sign of positive mating (or pregnant without sign of mating).

- Male mating: percent of males for which the female had sign of positive mating (or was pregnant without sign of mating) out of total males cohabited.

- Male fertility: percent of males for which the female was pregnant out of total males for which the female had sign of positive mating (or was pregnant without sign of mating).

- Parturition: percent of females with any pups (live or stillborn) out of total females with confirmed pregnancy.

- Gestation: percent of females with nay live pups out of total females with confirmed pregnancy.

Offspring viability indices:

- Live Birth: percent of pups born alive out of total pups born

- Viability: percent of pups alive on day 4 (pre-culling) out of total pups born alive

- Lactation: percent of pups alive on day 21 out of total pups alive on day 4 (post-culling)

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Description (incidence and severity):

No deaths in the P or F1 animals. No treatment related clinical signs were observed during the study.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Mean body weights of animals dosed at 8000 and 12000 ppm were lower than controls throughout the study period.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Mean body weights of animals dosed at 8000 and 12000 ppm were lower than controls throughout the study period.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Minimal hepatocellular hypertrophy was associated with liver weight changes.

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Test substance intake: Test material intake decreased with food intake in males during the study. In females, test material intake fluctuated with the stages of the study (premating, gestation and lactation)

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
There were no deaths and no treatment-related clinical signs among the P parental animals. Incidental clinical signs occasionally observed included: hair loss, wound, crust/scruff, mass, chromodacryorrhoea and distended abdomen. There were no deaths in the F1 parental animals. Incidental findings included hair loss, wound, crust/scruff, mass and chromodacryorrhoea. One male was observed to have ectopic testes (at 12000 ppm), this was not confirmed at necropsy and was considered to be incidental.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
Mean body weights of animals (P generation) dosed at 8000 and 12000 ppm were lower than controls throughout the study period. Mean bodyweight gains were also significantly lower than those of the controls for animals in these dose groups. Adult males were affected throughout the study period. Adult females were affected to a somewhat lesser degree, primarily during the premating phases, but also during gestation and lactation; the effects were only minimally apparent in the 8000 ppm females. Food consumption was reduced among males dosed at 8000 and 12000 ppm throughout the study, and to a lesser extent in females dosed at 12000 ppm particularly during the premating period.
The F1 generation males from the 500 and 1000 ppm were 1.9 and 2.4 days (respectively) older that the control groups, which is considered to account for the significantly mean higher starting bodyweights. These differences were not apparent at the end of weaning. The males from the 8000 and 12000 ppm groups were 1.5 and 2.2 day older respectively, the differences in bodyweight were apparent at the end of weaning. If all pups were of the same age, it was expected that the 500 and 1000 ppm animals would have been within the normal variation of bodyweight, in the 8000 and 12000 ppm groups, the weaning bodyweights would have been expected to be lower. At the beginning of the third pre-mating week mean bodyweights for the 8000 and 12000 ppm males were lower than that of the control animals, as was observed in the same dose groups of the P generation. Bodyweight gains for these groups remained depressed throughout the first 9 premating weeks. By the end of the premating, mean bodyweights were depressed in all treatment groups at termination of the study. By the fourth week of premating, the mean bodyweights of the 500 and 1000 ppm groups were not significantly different from the control groups. Initial mean bodyweights of the F1 females followed a similar pattern to the males. Mean bodyweights of the 500 and 1000 ppm females were higher than the controls and weights of the 8000 and 12000 ppm females were not lower than the controls. By the third premating week, bodyweights of 12000 ppm females were consistently lower than controls (statistically significant weeks 3 through 8 and the end of week 10). Bodyweight change was not affected by treatment during the premating period, however significantly lower bodyweight gains were noted for 1000 ppm females from weeks 1-2 and significantly higher gains for 1000, 8000 and 12000 ppm females from weeks 8-9 were not time or dose dependent. Mean bodyweights during the gestation days 7 and 14 were reduced in the 12000 ppm group. Bodyweight gains were not different among the groups during gestation. During the lactation period, the difference of the mean bodyweights of the controls compared to the 12000 ppm group decreased. Bodyweight gain in this group was positive during lactation, whereas all other groups lost weight during this week.

TEST SUBSTANCE INTAKE (PARENTAL ANIMALS)
Mean test item intake relative to bodyweight increased as nominal dietary concentration increased in roughly proportional patterns. In males, by the end of the 10 week pre-mating period, test item intake was approximately 50-60 % of the initial values. Relative test item intake continued to decrease slightly during the post-mating period. In females, by the end of the premating period, test item intake had reduced to approximately 60-70 % of the initial values as bodyweight increased. During the gestation period, test item intake increased by approximately 10 % above the initial premating values. Throughout the lactation period, test item intake was almost three times the value at the end of gestation. Test substance intake in the F1 generation followed a similar pattern to the P generation. Mean test item intake increased in proportionally as nominal dietary concentrations increased. In comparison to the P generation, test material intake relative to bodyweight at the beginning of F1 generation was substantially higher (26-53 % for males and 19-45 % for females), this is attributed to the younger age and lower bodyweight of the animals. During gestation, relative test item intake of dams in all groups increased during the first two weeks due to marginally higher food consumption, and then decreased during the third week in relation to the increased bodyweights. There was a marked increase (nearly 2-fold) increase over the lactation period due to increased food consumption.

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
The mean number of days in dams oestrous cycles did not differ among the groups in both the P and F1 generations and were within the normal range for rats. There were no obvious deviations from normality among the animals which were not pregnant.

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
Sperm morphology and motility was no affected by treatment with test material. There were no changes in the number of spermatids per gram of testis or in the number of spermatids per mg of cauda liquid.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
There were no treatment-related effects on the number of animals with evidence of positive mating and the number of females becoming pregnant in both the P and F1 generation. Successful mating generally occurred within one oestrous cycle in the P generation. Furthermore, there were no indications of effects of treatment with test material on any of the gestation or parturition indices. The mean length of gestation was similar among all groups.

ORGAN WEIGHTS (PARENTAL ANIMALS)
In P animals, absolute liver weights were not increased, but relative liver weights were increased in animals dosed at 8000 and 12000 ppm which may correspond to the minimal hepatocellular hypertrophy found on histopathological examination and are considered to be treatment-related. Mean absolute spleen weights were decreased and relative kidney and brain weights were increased at 8000 and 12000 ppm and adrenals, left testis and epididymides were increased at 12000 ppm. These differences are all attributed primarily to decreased terminal bodyweights.
In F1 animals, relative liver weights were increased in 8000 and 12000 ppm males as with the P animals this was considered to be related to the minimal hepatocellular hypertrophy found on histopathological examination. Mean absolute spleen weights were decreased in 8000 and 12000 ppm males. This decrease was attributed to reduced bodyweight. Both absolute and relative thymus weights were also decreased in these animals; these observations were also attributed to the lower bodyweights. All other findings in males were considered to be incidental.
In F1 females, increased relative liver weights in the 12000 ppm group was also considered to be related to the observed minimal hepatocellular hypertrophy. No such effects were noted in 8000 ppm females. Absolute spleen weights were decreased in 8000 and 12000 pm females and relative spleen weights were decreased at 8000 ppm. Absolute thymus weights were decreased in both the 8000 and 12000 ppm and relative spleen weights at 8000 ppm. Absolute thymus weights were decreased in both the 8000 and 12000 ppm groups. The lower spleen and thymus weights, as with the male animals were considered to be related to reduced bodyweight in these animals.

GROSS PATHOLOGY (PARENTAL ANIMALS)
No treatment-related macroscopic changes were observed at terminal necropsy of the P or F1 parental animals.

HISTOPATHOLOGY (PARENTAL ANIMALS)
No treatment-related microscopic changes in the reproductive organs were observed on histological examination from P and F1 males and females in the control and 12000 ppm groups, as well as in non-pregnant females and in males that failed to mate. Evaluation of sperm maturation in the testes of selected control and 12000 ppm males revealed no changes. There was an increased incidence of minimal hepatocellular hypertrophy in both sexes at 8000 and 12000 ppm in both generations. In the F1 males, hepatocellular hypertrophy was increased in the 80000 ppm groups, but not in the 8000 ppm females. These changes were attributed to treatment.

Effect levels (P0)

Dose descriptor:

NOAEL

Effect level:

1 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: equivalent to 48-137 mg/kg/day for males and 60-199 mg/kg/day for females. The grand mean test material intake at this dose level was 83.4 mg/kg/day.

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Sexual maturation:

effects observed, treatment-related

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

no effects observed

Histopathological findings:

no effects observed

Details on results (F1)

VIABILITY (OFFSPRING)
There were no treatment-related effects on any of the pup survival parameters in the F1 and F2 pups, and parental loss was not different among the groups, the mean litter size at birth were comparable in all dose groups and the mean numbers of live pups per litter did not differ among the groups at any time during the lactation period. Sex ratios of the pups on days 0 and 21 post partum did not differ substantially.

CLINICAL SIGNS (OFFSPRING)
There were no clinical signs in F1 and F2 pups which could be attributed to treatment. The few observations were common for rat pups (thin fur, bite wound, not suckled, subcutaneous haemorrhage, partly cannibalised) and distributed across dose groups.

BODY WEIGHT (OFFSPRING)
Mean pup weights at birth, on day 4 both before and after culling and on day 7 were similar in all groups in both F1 and F2 pups. In F1 pups, there were treatment-related reductions in bodyweight gain after day 7 in 8000 and 12000 ppm pups of both sexes. In F2 pups, there were treatment related reductions in bodyweight gain after day 4 in 12000 ppm pups of both sexes and after day 14 in 8000 ppm pups. In both F1 and F2 pups, differences in absolute bodyweights reached statistical significance by day 14 in 12000 pm animals and by day 21 in 8000 ppm animals. In the other test groups, mean pup bodyweights and mean pup bodyweight gain were similar to those of the control group throughout the lactation and post-weaning periods.

SEXUAL MATURATION (OFFSPRING)
There were no differences among the groups in the number of days before male pups showed balanopreputial separation, nor in the mean bodyweights at that time. For female pups, the number of days before vaginal patency was greater in 8000 and 12000 ppm pups than in control group pups. This delayed maturation is consistent with significantly reduced bodyweights in the two highest dose groups.

ORGAN WEIGHTS (OFFSPRING)
In F1 pups, mean absolute spleen weights were decreased in 8000 and 12000 ppm males and in 8000 ppm females. In F2 pups, mean absolute and relative spleen weights were reduced in both male and female pups at 8000 and 12000 ppm. Absolute thymus weights were decreased, and relative brain weights increased, in F1 pups of the two highest dose groups. In the F2 pups, absolute thymus weights were decreased in male and female pups at 12000 ppm. Mean brain weights were increased in F2 pups at both 8000 and 12000 ppm. The decreased spleen and thymus weights are attributed to delayed development in 8000 and 12000 ppm F1 and F2 pups, and the increased brain weight are consistent with significantly reduced bodyweights.

GROSS PATHOLOGY (OFFSPRING)
No treatment-related macroscopic findings were noted at necropsy of the F1 pups.

HISTOPATHOLOGY (OFFSPRING)
A few organ specific findings were noted in pups from both the F1 and F2 generation, but were not considered to be related to treatment.

Effect levels (F1)

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

1 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: equivalent to 48-137 mg/kg/day for males and 60-199 mg/kg/day for females. The grand mean test material intake at this dose level was 83.4 mg/kg/day.

Overall reproductive toxicity

Reproductive effects observed:

not specified

Any other information on results incl. tables

Table 2: Mean Bodyweights P Males (g)

Day	Dose level
	0 ppm	500 ppm	1000 ppm	8000 ppm	12000 ppm
1	188.8	185.2	185.4	185.2	183.7
8	245.7	237.6	239.0	231.6	230.5
15	297.6	286.0	289.2	278.1*	274.5*
22	333.2	323.1	323.1	311.0*	307.6**
29	365.6	351.6	353.4	337.8*	335.1**
36	391.8	376.4	376.6	360.1**	355.3**
43	413.4	397.7	397.2	378.3**	373.5**
50	430.6	414.6	410.6	393.6**	384.0**
57	444.8	428.0	423.2	405.3**	393.5**
64	459.2	442.3	435.1	418.0**	404.1**
68	470.6	452.2	445.1	427.4**	414.0**
71	462.3	444.3	435.2	416.9**	400.0**
78	467.0	453.5	450.2	427.7	414.8**
85	480.3	468.4	462.5	442.3**	426.9**
92	497.4	484.9	478.0	457.5*	439.5**
99	510.0	496.2	488.4	468.0*	448.6**
106	524.5	511.7	501.3	482.6*	461.7**
113	535.6	523.0	511.3	491.2*	468.2**
120	548.8	543.5	521.5	502.7*	479.9**

 * p≤0.05; ** p≤0.01

 

Table 3: Mean Bodyweights P Females (g)

Day		Dose level
		0 ppm	500 ppm	1000 ppm	8000 ppm	12000 ppm
Pre-mating	1	136.3	138.2	135.6	138.0	135.0
8		161.1	163.7	161.9	163.5	160.1
15		184.6	187.7	185.0	185.0	179.6
22		202.3	205.8	202.9	201.1	195.0
29		215.7	221.7	218.0	215.2	209.2
36		226.1	233.2	228.2	227.0	218.6
43		237.0	244.4	237.8	237.1	226.3
50		248.0	253.3	246.4	243.9	233.7*
57		251.9	258.4	251.2	247.8	237.7*
64		258.3	264.4	258.6	256.3	245.2*
68		260.4	286.6	261.0	259.0	248.0
Gestation	0	262.5	268.5	258.5	256.0	246.2*
7		285.1	294.1	282.9	277.9	267.9*
14		315.7	325.7	311.3	305.7	295.8**
21		398.6	411.1	392.5	386.4	374.6*
Lactation	0	297.4	305.0	294.2	283.0	276.1*
7		306.0	304.3	297.3	294.2	285.4**
14		320.8	322.4	315.3	308.7	303.4*
21		312.8	316.4	310.5	306.3	306.0

 * p≤0.05; ** p≤0.01

 

Table 4: Summary of Mating Parameters P Males and Females

	Dose level
	0 ppm	500 ppm	1000 ppm	8000 ppm	12000 ppm
Females placed with males	30	30	30	30	30
Total no inseminated	29	29	29	30	30
Female mating index (%)	96.7	96.7	96.7	100	100
Pregnant	27	27	27	29	28
Female fertility index (%)	93.1	93.1	93.1	96.7	93.3
Males placed with females	30	30	30	30	30
Mated	27	27	27	29	28
Male mating index (%)	96.7	96.7	96.7	100	100
with females pregnant	27	27	27	29	28
Male fertility index (%)	93.1	93.1	93.1	96.7	93.3
Females with defined day 0 pc	29	29	28	30	30
Mating days until day 0 pc	5.6	4.2	3.2	4.2	3.5

 

Table 5: Mean Bodyweights F1 Males (g)

Day	Dose level
	0 ppm	500 ppm	1000 ppm	8000 ppm	12000 ppm
134	149.4	166.3	169.1	146.9	145.3
141	205.3	222.0	222.8	193.6	191.1
148	258.1	274.5	276.8	239.4	235.7
155	306.6	319.8	324.2	281.7	279.2
162	344.7	355.3	360.8	313.0	309.3
169	377.5	385.0	390.1	339.8	334.3
176	402.4	410.1	414.0	361.4	355.1
183	423.0	430.4	432.5	379.4	371.7
190	440.1	447.2	450.0	394.8	385.0
197	456.4	463.7	464.6	407.6	395.3
201	464.8	473.1	472.9	416.1	405.3
204	458.7	466.9	465.7	410.3	398.5
211	467.8	475.2	475.8	421.6	409.9
218	483.5	488.4	492.6	432.5	419.2
225	497.2	503.1	508.7	445.1	433.8
232	505.7	511.1	517.0	452.1	440.0
239	519.1	526.6	528.9	465.2	454.1
246	529.3	539.3	541.8	475.7	462.6
253	538.3	549.8	549.1	481.7	470.7

 

Table 6: Mean Bodyweights F1 Females (g)

Day		Dose level
		0 ppm	500 ppm	1000 ppm	8000 ppm	12000 ppm
Premating	134	129.3	140.4	141.8	130.8	126.2
141		158.8	169.4	167.9	159.4	153.0
148		184.3	193.7	191.3	183.0	174.9
155		206.4	215.5	213.1	204.6	196.0
162		223.5	231.4	228.3	221.5	211.6
169		237.1	243.7	239.0	233.8	223.3
176		247.2	254.9	249.3	245.7	234.2
183		256.6	262.8	256.7	252.4	242.5
190		260.2	267.5	263.0	260.6	248.6
197		264.3	271.8	267.2	264.9	252.3
201		271.9	278.3	272.8	271.4	258.6
Gestation	0	270.1	275.8	271.8	268.0	258.6
7		294.9	300.4	294.3	288.7	279.5
14		323.9	328.5	324.0	318.1	305.2
21		406.2	415.4	409.0	400.2	385.0
Lactation	0	308.6	314.6	307.3	296.5	292.0
7		313.9	316.6	313.0	309.1	295.9
14		328.0	332.4	329.1	323.9	308.9
21		320.9	324.1	323.7	319.6	311.2

 

Table 7: Summary of Mating Parameters F1 Males and Females

	Dose level
	0 ppm	500 ppm	1000 ppm	8000 ppm	12000 ppm
Females placed with males	30	30	30	30	30
Total no inseminated	30	30	30	30	30
Female mating index (%)	100	100	100	100	100
Pregnant	28	26	26	26	29
Female fertility index (%)	93.3	86.7	86.7	86.7	96.7
Males placed with females	30	30	30	30	30
Mated	30	30	30	30	30
Male mating index (%)	100	100	100	100	100
with females pregnant	28	26	26	26	29
Male fertility index (%)	93.3	86.7	86.7	86.7	96.7
Females with defined day 0 pc	30	30	30	30	28
Mating days until day 0 pc	3.8	4.9	3.9	3.7	4.5

Table 8: Mean test item intake (mg/kg bodyweight/day) P and F1 generations

	Dose level (ppm)
	500		1000		8000		12000
	Begin	End	Begin	End	Begin	End	Begin	End
P Generation
Males
Premating	52	28	108	56	365	467	1144	728
Postmating	27	24	53	48	435	400	662	633
Females
Premating	50	33	105	65	768	539	1133	821
Gestation	37	35	75	72	628	583	917	875
Lactation	45	100	100	199	892	1548	1467	2374
F1 Generation
Males
Premating	70	29	137	55	1133	446	1755	736
Postmating	28	25	54	49	454	412	726	652
Females
Premating	66	32	125	60	1088	500	1645	792
Gestation	37	35	73	68	577	554	919	853
Lactation	48	97	98	199	876	1557	1161	2328

 

 

Applicant's summary and conclusion

Conclusions:

Under the conditions of the study, dietary administration of test material to rats through two generations produced effects on bodyweight and bodyweight gain, food consumption, organ weights and liver histopathology at dietary concentrations of 8000 and 12000 ppm. The findings were by and large consistent and indicative of generalised toxic properties of the test material.

Reduced bodyweight and bodyweight gain were apparent in adult males and females in the 8000 and 12000 ppm dose groups in both generations. Adult males were affected throughout the study period. Adult females were affected to a somewhat lesser extent, primarily during the premating phases, but also during gestation and lactation; the effects were only minimally apparent in 8000 ppm females.

Food consumption was also reduced among 8000 and 12000 ppm males throughout the study, and to a lesser extent in 12000 ppm females. Most organ weight changes considered to be affects of retarded growth/reduced feeding were also seen in the adult animals in these two dose groups.

The liver was identified as the target organ, based on findings of increased liver weights which correlated with minimal hepatocellular hypertrophy on adult males and females at the two highest feeding levels in both generations.

In pups, reduced bodyweight and bodyweight gain were seen at 8000 and 12000 ppm from the second lactation week onwards in both generations, and subsequent effects on organ weights were also observed.

The NOAEL was defined as 1000 ppm in both males and females which is approximately equal to 48-137 mg/kg/day for males and 60-199 mg/kg/day for females. The grand mean test material intake at this dose levels was 83.4 mg/kg/day.

There were no effects of dietary administration of the test material on any of the reproductive parameters assessed in this study.

Executive summary:

The reproductive toxicity of the test material was determined in accordance with standardised guidelines OECD 416, EPA OPP 83-4 and EPA OPPTS 870.3800. During the study rats were dosed test material in diet at 0, 500, 1000, 8000 and 12000 ppm. After 10 weeks of exposure, the animals (P) were cohabited 1:1 within each dose group for up to 21 days. Gestation and delivery were allowed to occur naturally. Litters (F1) were culled to four males and four females on day 4 post partum. After weaning of the last litter, selected F1 young (30 animals per sex and dose) were continuously exposed to test material for 10 weeks after which time animals of the same group were cohabited 1:1 for up to 21 days. Gestation and delivery were allowed to occur naturally. Resulting F2 litters were necropsied after weaning. Clinical observations, bodyweights, food consumption, mating, gestation and delivery parameters, oestrous cycle stages and sperm characteristics, pup survival, pup developmental landmarks, pup sexual maturation (F1), necropsy examinations of adults and pups and histopathological observations in sex and target organs of mating were recorded.

Under the conditions of the study, dietary administration of test material to rats through two generations produced effects on bodyweight and bodyweight gain, food consumption, organ weights and liver histopathology at dietary concentrations of 8000 and 12000 ppm. The findings were by and large consistent and indicative of generalised toxic properties of the test material. Reduced bodyweight and bodyweight gain were apparent in adult males and females in the 8000 and 12000 ppm dose groups in both generations. Adult males were affected throughout the study period. Adult females were affected to a somewhat lesser extent, primarily during the premating phases, but also during gestation and lactation; the effects were only minimally apparent in 8000 ppm females. Food consumption was also reduced among 8000 and 12000 ppm males throughout the study, and to a lesser extent in 12000 ppm females. Most organ weight changes considered to be effects of retarded growth/reduced feeding were also seen in the adult animals in these two dose groups.

The liver was identified as the target organ, based on findings of increased liver weights which correlated with minimal hepatocellular hypertrophy on adult males and females at the two highest feeding levels in both generations.

In pups, reduced bodyweight and bodyweight gain were seen at 8000 and 12000 ppm from the second lactation week onwards in both generations, and subsequent effects on organ weights were also observed.

The NOAEL was defined as 1000 ppm in both males and females which is approximately equal to 48-137 mg/kg/day for males and 60-199 mg/kg/day for females. The grand mean test material intake at this dose levels was 83.4 mg/kg/day.

There were no effects of dietary administration of the test material on any of the reproductive parameters assessed in this study.