Orthosulfamuron

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

In order to determine the maximum tolerated dietary concentration of IR5878 suitable for use in a subsequent two-generation study, IR5878 was administered in the diet at target concentrations of 14000, 7000 and 1400 ppm during pre-pairing, pairing and gestation periods and at reduced target concentrations of 9000, 4500 andfemales during lactation and after weaning.

The lowest NOAEL (No Observed Adverse Effect Level) of the study was based on microscopic findings on the liver and was set at 1400/900 ppm. Based on the data it was concluded that 350/225, 1400/900 and 5600/3600 ppm would be suitable dietary concentrations of IR5878 in the subsequent two-generation study.

A study was conducted to provide general information concerning the effects of IR5878 on reproductive function as assessed by gonadal function, estrous cycles, mating behaviour, conception, parturition, lactation, weaning and the growth and development of the off-spring. The study had the purpose to provide also information about the effects of IR5878 on neonatal morbidity, mortality, development and behaviour. Groups of 24 male and 24 female HanBrl: WIST rats received IR5878 at the doses of 0, 350, 1400 and 5600 ppm in their diet for 10 consecutive weeks and then mated. Litters derived from mating of F0 generation were utilised to form the basis of the F1 generation (24 males and 24 females per group). The mating of F1 animals was performed after at least 120 days of treatment. Concentrations of IR5878 in the diet were reduced to 0, 225, 900 and 3600 ppm in order to achieve a constant intake in terms of mg/kg/day and avoid over dosage during lactation of F0 and F1 dams and the rearing of freshly weaned pups.

The overall NOAEL in adult animals (F0 and F1 generation) was established at 1400/900 ppm, based on increased liver and kidney weights at 5600 ppm (F0 males only) and the existence of histopathological changes in liver and kidneys at 5600/3600 ppm in both generations and sexes. The NOAEL for reproductive effects was established at 5600/3600 ppm based on the absence of adverse reproductive effect in both generations. The NOAEL pup development was established at 5600/3600 ppm, on the basis of absence of any developmental effects in both pup generations. The NOAEL pup behaviour was established at 1400/900 ppm, based on reduced locomotor activity in F1 male pups at 5600/3600 ppm.

Link to relevant study records

Reference

Reference 1

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

2002

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

test procedure in accordance with national standard methods with acceptable restrictions

Qualifier:

no guideline followed

Principles of method if other than guideline:

The purpose of the study was to determine the maximum tolerated dietary concentration of IR5878 suitable for use in a subsequent two-generation study.
IR5878 was administered in the diet at target concentrations of 14000, 7000 and 1400 ppm during pre-pairing, pairing and gestation periods and at reduced target concentrations of 9000, 4500 and 900 in females during lactation and after weaning. Due to the loss of IR5878 during pelleting, achieved IR5878 concentrations in the diet were lower than target concentrations.

GLP compliance:

no

Specific details on test material used for the study:

Test material:
IR5878
Batch number: FCF/T/200-01 (ex 20525/03/2)
Purity: 98.44 %

Species:

rat

Strain:

other: HanBrl: WIST (SPF)

Sex:

male/female

Details on test animals or test system and environmental conditions:

Test animals:
Species and Strain: Male and Female HanBrl: WIST (SPF) rats
Age: 8 weeks minimum
Body weight: 271 ÷ 307 g (males); 168 ÷ 198 g (females)
Housing: individually in Makrolon cages type-3 with wire mesh tops. During the pairing period rats were housed one male/one female in Makrolon pairing cages.
Feed: pelleted standard Kliba 3433 rat/mouse maintenance diet ad libitum. Tap water ad libitum.

Environmental conditions:
Temperature and humidity measured during the study were 22 ± 3°C and 30 ÷ 70%, respectively.
Light: artificial fluorescent light with a 12-hour cycle.

Route of administration:

oral: feed

Details on exposure:

IR5878 was administered in the diet at target concentrations of 14000, 7000 and 1400 ppm during pre-pairing, pairing and gestation periods and at reduced target concentrations of 9000, 4500 and 900 in females during lactation and after weaning. Due to the loss of IR5878 during pelleting, achieved IR5878 concentrations in the diet were lower than target concentrations.

Details on mating procedure:

During the pairing period rats were housed one male/one female in Makrolon pairing cages.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Due to loss of the test item during pelleting, achieved test item concentrations in the diet were lower than target concentrations.

Duration of treatment / exposure:

In order to determine the maximum tolerated dietary concentration of IR5878 suitable for use in a subsequent two-generation study, IR5878 was administered in the diet at target concentrations of 14000, 7000 and 1400 ppm during pre-pairing, pairing and gestation periods and at reduced target concentrations of 9000, 4500 and 900 in females during lactation and after weaning.

Frequency of treatment:

In order to determine the maximum tolerated dietary concentration of IR5878 suitable for use in a subsequent two-generation study, IR5878 was administered in the diet at target concentrations of 14000, 7000 and 1400 ppm during pre-pairing, pairing and gestation periods and at reduced target concentrations of 9000, 4500 and 900 in females during lactation and after weaning.

Dose / conc.:

1 400 ppm

Dose / conc.:

4 500 ppm

Dose / conc.:

9 000 ppm

No. of animals per sex per dose:

IR5878 was administered in the diet to males and females at target concentrations of 14000, 7000 and 1400 ppm during pre-pairing, pairing and gestation periods and at reduced target concentrations of 9000, 4500 and 900 in females during lactation and after weaning.

Control animals:

yes, concurrent vehicle

Details on study design:

The purpose of the study was to determine the maximum tolerated dietary concentration of IR5878 suitable for use in a subsequent two-generation study.
IR5878 was administered in the diet at target concentrations of 14000, 7000 and 1400 ppm during pre-pairing, pairing and gestation periods and at reduced target concentrations of 9000, 4500 and 900 in females during lactation and after weaning. Due to the loss of IR5878 during pelleting, achieved IR5878 concentrations in the diet were lower than target concentrations.

Parental animals: Observations and examinations:

General tolerability
No mortalities or clinical signs were noted in any animal.
At 14000/9000 ppm, reduced food consumption was noted during the first week of administration in both males and females. Also at 7000/4500 ppm mean food consumption of females was slightly reduced during the first week of the pre-pairing period. After this initial reduction, food consumption in males and females at all dosages was similar to that of vehicle controls.
No differences between groups, that were considered to be test item related, were noted in female or male body weight development.

Oestrous cyclicity (parental animals):

There was no test item-related effect on fertility, mating performance, duration of gestation.

Sperm parameters (parental animals):

There was no test item-related effect on fertility, mating performance, duration of gestation.

Litter observations:

Mean litter size at first litter check was similar in all groups. One dead pup was noted at 1400/900 ppm and 14000/9000 ppm each, but this low incidence was considered to be incidental.
At 7000/4500 ppm and 14000/9000 ppm, one dam lost one pup between days 0 and 4 post partum.. Also at 7000/4500 ppm and 14000/9000 ppm, one dam lost one pup between days 5 and 21 post partum. Due to the low number of litters it cannot be concluded whether these losses were test item-related. However, this incidence of post natal and breeding loss is quite common and could therefore very well be incidental.

Postmortem examinations (parental animals):

Terminal examinations
No abnormal findings were noted during macroscopical examination of parent animals.
At 14000/9000 ppm and at 7000/4500 ppm, a centrilobular, small vacuolar fatty change along with a hepatocellular hypertrophy (zone 3 to diffuse) was recorded in the liver. Additionally, compared to controls, a decrease in glycogen content was obvious.
At 1400/900 ppm a slight hepatocellular hypertrophy was noted in one male and one female.
In the kidney, an increased incidence in tubular basophilia was recorded in animals at 1400/900 ppm, which is indicative for increased tubular degeneration/regeneration.

Postmortem examinations (offspring):

No macroscopic findings that were considered to be test item-related were noted at necroscopy of pups.

Reproductive indices:

Also, the mean number of implantations per dam and post-implantation loss were similar in all groups and gave no indication for any test item-related effects.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

No mortalities or clinical signs were noted in any animal.
At 14000/9000 ppm, reduced food consumption was noted during the first week of administration in both males and females. Also at 7000/4500 ppm mean food consumption of females was slightly reduced during the first week of the pre-pairing period. After this initial reduction, food consumption in males and females at all dosages was similar to that of vehicle controls.
No differences between groups, that were considered to be test item related, were noted in female or male body weight development.

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

At 14000/9000 ppm, reduced food consumption was noted during the first week of administration in both males and females. Also at 7000/4500 ppm mean food consumption of females was slightly reduced during the first week of the pre-pairing period. After this initial reduction, food consumption in males and females at all dosages was similar to that of vehicle controls.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

At 14000/9000 ppm and at 7000/4500 ppm, a centrilobular, small vacuolar fatty change along with a hepatocellular hypertrophy (zone 3 to diffuse) was recorded in the liver. Additionally, compared to controls, a decrease in glycogen content was obvious.
At 1400/900 ppm a slight hepatocellular hypertrophy was noted in one male and one female.
In the kidney, an increased incidence in tubular basophilia was recorded in animals at 1400/900 ppm, which is indicative for increased tubular degeneration/regeneration.

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

There was no test item-related effect on fertility, mating performance, duration of gestation.
Also, the mean number of implantations per dam and post-implantation loss were similar in all groups and gave no indication for any test item-related effects.

Clinical signs:

no effects observed

Mortality:

mortality observed, non-treatment-related

Description (incidence):

Mean litter size at first litter check was similar in all groups. One dead pup was noted at 1400/900 ppm and 14000/9000 ppm each, but this low incidence was considered to be incidental.
At 7000/4500 ppm and 14000/9000 ppm, one dam lost one pup between days 0 and 4 post partum.. Also at 7000/4500 ppm and 14000/9000 ppm, one dam lost one pup between days 5 and 21 post partum. Due to the low number of litters it cannot be concluded whether these losses were test item-related. However, this incidence of post natal and breeding loss is quite common and could therefore very well be incidental.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Also, there was no test item-related effect on mean pup weight at birth. Slightly increased mean pup weights at 1400/900 ppm and 7000/4500 ppm (5.9 g in both groups compared to 5.5 g in vehicle controls, combined data for male and female pups) were probably due to the smaller litter size in these groups. Mean pup weight at 14000/9000 ppm was similar to vehicle controls (5.6 g).
Also on day 21 post partum, mean pup weights were similar in all groups and therefore gave no indication for any test item-related effect.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Reared beyond weaning
During the one week rearing period beyond weaning, no adverse effects on mean food consumption or body weight development were noted.

Gross pathological findings:

no effects observed

Description (incidence and severity):

Terminal examinations
No macroscopic findings that were considered to be test item-related were noted at necroscopy of pups.

Conclusions:

The lowest NOAEL (No Observed Adverse Effect Level) of the study was based on microscopic findings on the liver and was set at 1400/900 ppm.
Based on the data it was concluded that 350/225, 1400/900 and 5600/3600 ppm would be suitable dietary concentrations of IR5878 in the subsequent two-generation study.

Executive summary:

In order to determine the maximum tolerated dietary concentration of IR5878 suitable for use in a subsequent two-generation study, IR5878 was administered in the diet at target concentrations of 14000, 7000 and 1400 ppm during pre-pairing, pairing and gestation periods and at reduced target concentrations of 9000, 4500 andfemales during lactation and after weaning. No abnormal findings were noted at first litter check or during lactation.

The lowest NOAEL (No Observed Adverse Effect Level) of the study was based on microscopic findings on the liver and was set at 1400/900 ppm. Based on the data it was concluded that 350/225, 1400/900 and 5600/3600 ppm would be suitable dietary concentrations of IR5878 in the subsequent two-generation study.

Reference 2

Endpoint:

two-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2003

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3800 (Reproduction and Fertility Effects)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Test material:
IR5878
Batch number: G 009/02
Purity: 98.56 ± 0.19 %

Species:

rat

Strain:

other: HanBrl: WIST (SPF)

Details on species / strain selection:

Species and Strain: Male and Female HanBrl: WIST (SPF) rats

Sex:

male/female

Details on test animals or test system and environmental conditions:

Test animals:
Species and Strain: Male and Female HanBrl: WIST (SPF) rats
Age: 6 weeks minimum
Body weight: 184 ÷ 227 g (males); 131 ÷ 167 g (females)
Housing: individually in Makrolon cages type-3 with wire mesh tops. During the pairing period rats were housed one male/one female in Makrolon pairing cages.
Feed: granulated standard Kliba 3433 rat/mouse maintenance diet ad libitum. Community tap water ad libitum.

Environmental conditions:
Temperature and humidity measured during the study were 22 ± 3°C and 30 ÷ 70%, respectively.
Light: artificial fluorescent light with a 12-hour cycle.

Route of administration:

oral: feed

Details on exposure:

Groups of 24 male and 24 female HanBrl: WIST rats received IR5878 at the doses of 0, 350, 1400 and 5600 ppm in their diet for 10 consecutive weeks and then mated.

Details on mating procedure:

Litters derived from mating of F0 generation were utilised to form the basis of the F1 generation (24 males and 24 females per group). The mating of F1 animals was performed after at least 120 days of treatment.

Analytical verification of doses or concentrations:

no

Details on analytical verification of doses or concentrations:

Mean test item intakes
The high food spillage due to the powder diet determined a test item intake unrealistically high. Therefore IR5878 intake was calculated with the mean values obtained in another concurrent 2-generation study performed with same rat strain, and same kind of diet, but pelleted (see point IIA 05.05.01/01).

Duration of treatment / exposure:

Groups of 24 male and 24 female HanBrl: WIST rats received IR5878 at the doses of 0, 350, 1400 and 5600 ppm in their diet for 10 consecutive weeks and then mated. ). The mating of F1 animals was performed after at least 120 days of treatment. Concentrations of IR5878 in the diet were reduced to 0, 225, 900 and 3600 ppm in order to achieve a constant intake in terms of mg/kg/day and avoid over dosage during lactation of F0 and F1 dams and the rearing of freshly weaned pups.

Frequency of treatment:

Groups of 24 male and 24 female HanBrl: WIST rats received IR5878 at the doses of 0, 350, 1400 and 5600 ppm in their diet for 10 consecutive weeks and then mated. ). The mating of F1 animals was performed after at least 120 days of treatment. Concentrations of IR5878 in the diet were reduced to 0, 225, 900 and 3600 ppm in order to achieve a constant intake in terms of mg/kg/day and avoid over dosage during lactation of F0 and F1 dams and the rearing of freshly weaned pups.

Dose / conc.:

0 ppm (nominal)

Dose / conc.:

350 ppm (nominal)

Dose / conc.:

1 400 ppm (nominal)

Dose / conc.:

5 600 ppm (nominal)

No. of animals per sex per dose:

Groups of 24 male and 24 female

Parental animals: Observations and examinations:

The purpose of the study was to provide general information concerning the effects of IR5878 on reproductive function as assessed by gonadal function, estrous cycles, mating behaviour, conception, parturition, lactation, weaning and the growth and development of the off-spring. The study had the purpose to provide also information about the effects of IR5878 on neonatal morbidity, mortality, development and behaviour.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Clinical signs and mortality
Treatment with IR5878 was generally well tolerated and no test item-related mortalities, no signs of discomfort or clinical signs of reaction to treatment were observed in any group.
One F0 female of the highest group was killed in extremis on day 15 of prepairing period (the cause of morbidity was a congenital internal hydrocephalus). One F1 female of the highest group was found died on day 89 of prepairing period (the cause of mortality was a pyometra). These isolated mortalities were considered to be incidental.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

Clinical signs and mortality
Treatment with IR5878 was generally well tolerated and no test item-related mortalities, no signs of discomfort or clinical signs of reaction to treatment were observed in any group.
One F0 female of the highest group was killed in extremis on day 15 of prepairing period (the cause of morbidity was a congenital internal hydrocephalus). One F1 female of the highest group was found died on day 89 of prepairing period (the cause of mortality was a pyometra). These isolated mortalities were considered to be incidental.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Mean body weight in both sexes and in both parent generations did not give any indication for treatment-related effects. The few and occasional differences observed between treated animals and controls were considered incidental and not to be of toxicological relevance.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

All food consumption values of 50 g/animal/day or higher were excluded from the calculation of food consumption as being obviously caused by food spillage. The food spillage is a common phenomenon during studies with unpelleted diet.
Mean food consumption of F0 and F1 animals at all dosages was not affected by treatment with IR5878.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

Liver: minimal multifocal, small-vacuolar fatty degeneration was recorded in one male F0 animal treated at 5600 ppm. This change was considered as an adverse effect. Minimal to slight hepatocellular hypertrophy (zone 3 to diffuse) was observed in males and females of both generations at 5600 ppm. This change was regarded as an adaptive change.Kidney: an increased incidence and mean grade of urothelial hyperplasia was recorded in males and females of both generations treated at 5600 ppm, but the relevance of this finding in respect of IR5878 relationship is unclear.

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

No difference between test item-treated and control animals was detected in reproductive organs.

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

No treatment-related effects on sperm motility were noted in both generations at any dose level.
The statistically significant variations in progressively motile and stationary motile sperm observed in F0 treated at 5600 ppm was considered incidental, because the overall number of motile sperm was similar to control.
Analysis of sperm morphology in both generations (performed only in the control and in the highest groups) gave no indication for any IR5878 related effects.
Determination of homogenisations-resistant spermatids obtained from caudal epididymides or testes tissue samples performed in the control and in the highest groups of both generations gave no indication for any test item-related effects.

Reproductive performance:

no effects observed

For both generations and at all dietary concentrations there were no treatment-related effects on estrous cycles, median precoital time, fertility indices, mean duration of gestation, number of implantations, post-implantation loss, pup survival or litter size from birth through to weaning.

Key result

Dose descriptor:

NOAEL

Effect level:

>= 1 400 ppm

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

organ weights and organ / body weight ratios

histopathology: non-neoplastic

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Clinical signs and mortality
Treatment with IR5878 was generally well tolerated and no test item-related mortalities, no signs of discomfort or clinical signs of reaction to treatment were observed in any group.
One F0 female of the highest group was killed in extremis on day 15 of prepairing period (the cause of morbidity was a congenital internal hydrocephalus). One F1 female of the highest group was found died on day 89 of prepairing period (the cause of mortality was a pyometra). These isolated mortalities were considered to be incidental.

Mortality:

mortality observed, non-treatment-related

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Mean body weight in both sexes and in both parent generations did not give any indication for treatment-related effects. The few and occasional differences observed between treated animals and controls were considered incidental and not to be of toxicological relevance.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

All food consumption values of 50 g/animal/day or higher were excluded from the calculation of food consumption as being obviously caused by food spillage. The food spillage is a common phenomenon during studies with unpelleted diet.
Mean food consumption of F0 and F1 animals at all dosages was not affected by treatment with IR5878.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Test item-related effects on organ weight were confined to increase liver and kidney weights in F0 males treated at 5600 ppm. The increase of the weight of brain, pituitary, thyroid and testes were considered not to be related to treatment with IR5878. The organ weights in F0 females and in F1 males and females were not considered to be affected by treatment with IR5878. The marginal differences noted were considered to be of no toxicological relevance and to reflect normal biological variability.

Gross pathological findings:

no effects observed

Description (incidence and severity):

All gross necropsy findings in both parental generations were within the range of spontaneous lesions in rats of this strain and age. Their inter-group distribution did not suggest an effect of treatment with IR5878 at any dose level.

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

Liver: minimal multifocal, small-vacuolar fatty degeneration was recorded in one male F0 animal treated at 5600 ppm. This change was considered as an adverse effect. Minimal to slight hepatocellular hypertrophy (zone 3 to diffuse) was observed in males and females of both generations at 5600 ppm. This change was regarded as an adaptive change.
Kidney: an increased incidence and mean grade of urothelial hyperplasia was recorded in males and females of both generations treated at 5600 ppm, but the relevance of this finding in respect of IR5878 relationship is unclear.
No difference between test item-treated and control animals was detected in reproductive organs.
Quantitative ovarian histopathology data (only in F1 females)
Counts of ovarian follicles and corpora lutea were similar in Control and in high dose females and without any statistically significant difference There was no indication for IR5878 related effects on ovarian follicles in the F1 generation.

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

For both generations and at all dietary concentrations there were no treatment-related effects on estrous cycles, median precoital time, fertility indices, mean duration of gestation, number of implantations, post-implantation loss, pup survival or litter size from birth through to weaning.

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

No treatment-related effects on sperm motility were noted in both generations at any dose level.

Reproductive performance:

no effects observed

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

No findings considered to be test item related were noted at first litter check or during lactation.
Sex ratios (at first litter check and on day 21 post partum), pup weights (on day 1 to day 21 post partum), sexual maturation (recorded only for F1 generation) and modified Irwin Screen (performed only in F1) were unaffected by treatment at all dietary concentrations.
Locomotor activity (performed only for F1), as assessed quantitatively in terms of low beam counts in an activity monitor, was reduced in male F1 pups at 5600/3600 ppm.

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

Organ weight
Brain, thymus and spleen weights of F1 and F2 pups gave no indication of test item-related effects.

Gross pathological findings:

no effects observed

Description (incidence and severity):

The types and frequencies of gross lesions noted in F1 and F2 pups gave no indication of test item-related effects.

Histopathological findings:

no effects observed

Description (incidence and severity):

The types and frequencies of gross lesions noted in F1 and F2 pups gave no indication of test item-related effects.

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

>= 1 400 ppm

Based on:

act. ingr.

Sex:

male

Basis for effect level:

clinical signs

Key result

Reproductive effects observed:

no

Lowest effective dose / conc.:

5 600 ppm (nominal)

Treatment related:

yes

Findings

Mean test item intakes

The high food spillage due to the powder diet determined a test item intake unrealistically high. Therefore IR5878 intake was calculated with the mean values obtained in another concurrent 2-generation study performed with same rat strain, and same kind of diet, but pelleted (see point IIA 05.05.01/01).

 

Table 5.6-2:    Mean test item intakes achieved for all animals at each period of treatment (mg/kg/day)

			Group 350/225 ppm	Group 1400/900 ppm	Group 5600/3600 ppm
F0	Males	Prepairing	22.2	88.6	354.5
		After pairing	17.7	70.8	283.4
	Females	Prepairing	25.6	102.2	408.8
		Gestation	21.8	87.2	348.9
		Lactation	34.0	135.8	543.2
F1	Males	Prepairing	24.3	97.0	388.1
		After pairing	17.8	71.1	284.5
	Females	Prepairing	27.8	111.2	444.6
		Gestation	23.3	93.1	372.4
		Lactation	34.2	137.0	547.9

 

PARENTS ANIMALS

Clinical signs and mortality

Treatment with IR5878 was generally well tolerated and no test item-related mortalities, no signs of discomfort or clinical signs of reaction to treatment were observed in any group.

One F0 female of the highest group was killed in extremis on day 15 of prepairing period (the cause of morbidity was a congenital internal hydrocephalus). One F1 female of the highest group was found died on day 89 of prepairing period (the cause of mortality was a pyometra). These isolated mortalities were considered to be incidental.

 

Body weight and food consumption

Mean body weight in both sexes and in both parent generations did not give any indication for treatment-related effects. The few and occasional differences observed between treated animals and controls were considered incidental and not to be of toxicological relevance.

All food consumption values of 50 g/animal/day or higher were excluded from the calculation of food consumption as being obviously caused by food spillage. The food spillage is a common phenomenon during studies with unpelleted diet.

Mean food consumption of F0 and F1 animals at all dosages was not affected by treatment with IR5878.

 

Reproduction data

For both generations and at all dietary concentrations there were no treatment-related effects on estrous cycles, median precoital time, fertility indices, mean duration of gestation, number of implantations, post-implantation loss, pup survival or litter size from birth through to weaning.

 

Table 5.6-3:    Summary of reproductive performance – F0 generation

Dosage Findings	0 ppm	350/225 ppm	1400/900 ppm	5600/3600 ppm
Initial group size	24	24	24	24
Mortalities	-	-	-	1
Mean estrous cycles duration (days)	4.2	4.2	4.1	4.1
Number males/females paired	24/24	24/24	24/24	23/23
Number females mated	24	24	24	23
Median precoital time (days)	4	8	4	3
Number of pregnant females	23	23	22	22
Fertility index (%)	95.8	95.8	91.8	95.7
Duration of gestation (days)	21.8	21.6	21.6	21.7
Mean implantations for litter	12.2	12.3	12.6	11.7
Post-implantation loss (%)	4.3	4.3	2.9	4.3
Number of female bearing young	23	23	22	22
Mean living pups at first litter check	11.7	11.7	12.2	11.2
Gestation index (%)	100	100	100	100
Mean post-natal loss days 0-4 p.p.             (% living pups)	0.1 (1.1)	0.3 (2.2)	0.3 (2.6)	0.1 (1.2)
Mean breeding loss days 5-21 p.p.	0.0	0.0	0.1	0.0

Fertility index = (Females achieving a pregnancy/ females paried) x 100

Post-implantation loss = expressed as a percentage of total implantations

Gestation index = (No. of females with living pups/ No. of females pregnant) x 100

Mean postnatal loss days 0-4 p.p. = determined before culling

 

Table 5.6-4:    Summary of reproductive performance – F1 generation

Dosage Findings	0 ppm	350/225 ppm	1400/900 ppm	5600/3600 ppm
Initial group size	24	24	24	24
Mortalities	-	-	-	1
Mean estrous cycles duration (days)	4	4.0	4.1	4.1
Number males/females paired	24/24	23/23 *	24/24	23/23
Number females mated	24	21	23	23
Median precoital time (days)	2	4	2	3
Number of pregnant females	22	20	20	23
Fertility index (%)	91.7	87.0	83.3	100.0
Duration of gestation (days)	21.8	21.7	21.7	21.8
Mean implantations for litter	12.7	12.1	12.9	12.0
Post-implantation loss (%)	4.5	5.8	4.7	4.7
Number of female bearing young	21	20	20	23
Mean living pups at first litter check	12.5	11.4	12.3	11.4
Gestation index (%)	100	100	100	100
Mean post-natal loss days 0-4 p.p.             (% living pups)	0.0 (0.4)	0.3 (2.2)	0.4 ** (3.3)	0.1 (0.8)
Mean breeding loss days 5-21 p.p.	1.2	2.5 ***	1.5	2.0 *

Fertility index = (Females achieving a pregnancy/ females paried) x 100

Post-implantation loss = expressed as a percentage of total implantations

* = at selection of F1 animals for further rearing in group 2 (350 ppm), a male pup has erroneously been selected instead of female. This error did not become obvious before examination for vaginal opening began

Gestation index = (No. of females with living pups/ No. of females pregnant) x 100

Mean postnatal loss days 0-4 p.p. = determined before culling

** = Fisher’s exact test significant at 5% level

*** = Fisher’s exact test significant at 1% level

 

Macroscopic pathology

All gross necropsy findings in both parental generations were within the range of spontaneous lesions in rats of this strain and age. Their inter-group distribution did not suggest an effect of treatment with IR5878 at any dose level.

 

Organ weight data

Test item-related effects on organ weight were confined to increase liver and kidney weights in F0 males treated at 5600 ppm. The increase of the weight of brain, pituitary, thyroid and testes were considered not to be related to treatment with IR5878.

 

Table 5.6-5:    Organ weights F0 male animals

Dosage (ppm) Weights		0	350/225	1400/900	5600/3600
Body weight (g)^&		435.9	438.3	445.1	456.0
Liver	absolute (g)	13.5	13.33	13.48	15.24^**
	relative (%)	3.02	3.04	3.03	3.34^**
Kidneys	absolute (g)	2.27	2.34	2.26	2.48^**
	relative (%)	0.52	0.53	0.51	0.54
^&=body weighs were recorded on the day of necropsy ^**=Dunnett-test based on pooled variance sig. at 1% level

 

The organ weights in F0 females and in F1 males and females were not considered to be affected by treatment with IR5878. The marginal differences noted were considered to be of no toxicological relevance and to reflect normal biological variability.

 

Sperm analysis data

No treatment-related effects on sperm motility were noted in both generations at any dose level.

The statistically significant variations in progressively motile and stationary motile sperm observed in F0 treated at 5600 ppm was considered incidental, because the overall number of motile sperm was similar to control.

 

Table 5.6-6:    Sperm analysis F0 animals – motility

Dosage (ppm) Sperm motility	0	350/225	1400/900	5600/3600
% No motility	15	11	14	14
% Progressively motility	31	31	37	41^**
% Stationary motility	54	58	49	45^*
% Overall number motility sperm	85	89	86	86
^*/^**=Dunnett-test based on pooled variance sig. at 5% or 1% level

 

Analysis of sperm morphology in both generations (performed only in the control and in the highest groups) gave no indication for any IR5878 related effects.

Determination of homogenisations-resistant spermatids obtained from caudal epididymides or testes tissue samples performed in the control and in the highest groups of both generations gave no indication for any test item-related effects.

 

Histopathology data

Liver: minimal multifocal, small-vacuolar fatty degeneration was recorded in one male F0 animal treated at 5600 ppm. This change was considered as an adverse effect. Minimal to slight hepatocellular hypertrophy (zone 3 to diffuse) was observed in males and females of both generations at 5600 ppm. This change was regarded as an adaptive change.

Kidney: an increased incidence and mean grade of urothelial hyperplasia was recorded in males and females of both generations treated at 5600 ppm, but the relevance of this finding in respect of IR5878 relationship is unclear.

No difference between test item-treated and control animals was detected in reproductive organs.

 

Quantitative ovarian histopathology data (only in F1 females)

Counts of ovarian follicles and corpora lutea were similar in Control and in high dose females and without any statistically significant difference There was no indication for IR5878 related effects on ovarian follicles in the F1 generation.

 

PUPS DATA

Litter data (in both generations)

No findings considered to be test item related were noted at first litter check or during lactation.

Sex ratios (at first litter check and on day 21 post partum), pup weights (on day 1 to day 21 post partum), sexual maturation (recorded only for F1 generation) and modified Irwin Screen (performed only in F1) were unaffected by treatment at all dietary concentrations.

Locomotor activity (performed only for F1), as assessed quantitatively in terms of low beam counts in an activity monitor, was reduced in male F1 pups at 5600/3600 ppm.

 

Table 5.6-7:    Litter data (F1 pups)

Dosage Findings	0 ppm	350/225 ppm	1400/900 ppm	5600/3600 ppm
Sex ratios (% of males/females)	52/48	50/50	51/49	51/49
Body weight gain day 1 to 21 p.p. (g)	41.6	41.7	41.5	41.2
Preputial separation (days)	27.4	27.3	27.9	27.3
Vaginal patency (days)	34.3	34.7	34.5	34.4
Locomotor activity (low beams count)	2597	3018	2544	697 *

* =Dunnet –test based on pooled variance sig. at 1% level

 

Table 5.6-8:    Litter data (F2 pups)

Dosage Findings	0 ppm	350/225 ppm	1400/900 ppm	5600/3600 ppm
Sex ratios (% of males/females)	50/50	48/52	51/49	49/51
Body weight gain day 1 to 21 p.p. (g)	43.0	42.2	44.7	43.7

 

Macroscopic pathology and histopathology data

The types and frequencies of gross lesions noted in F1 and F2 pups gave no indication of test item-related effects.

 

Organ weight

Brain, thymus and spleen weights of F1 and F2 pups gave no indication of test item-related effects.

Conclusions:

The overall NOAEL in adult animals (F0 and F1 generation) was established at 1400/900 ppm, based on increased liver and kidney weights at 5600 ppm (F0 males only) and the existence of histopathological changes in liver and kidneys at 5600/3600 ppm in both generations and sexes.
The NOAEL for reproductive effects was established at 5600/3600 ppm based on the absence of adverse reproductive effect in both generations.
The NOAEL pup development was established at 5600/3600 ppm, on the basis of absence of any developmental effects in both pup generations.
The NOAEL pup behaviour was established at 1400/900 ppm, based on reduced locomotor activity in F1 male pups at 5600/3600 ppm.
No findings considered to be test item related were noted at first litter check or during lactation.

Executive summary:

The purpose of the study was to provide general information concerning the effects of IR5878 on reproductive function as assessed by gonadal function, estrous cycles, mating behaviour, conception, parturition, lactation, weaning and the growth and development of the off-spring. The study had the purpose to provide also information about the effects of IR5878 on neonatal morbidity, mortality, development and behaviour. Groups of 24 male and 24 female HanBrl: WIST rats received IR5878 at the doses of 0, 350, 1400 and 5600 ppm in their diet for 10 consecutive weeks and then mated. Litters derived from mating of F0 generation were utilised to form the basis of the F1 generation (24 males and 24 females per group). The mating of F1 animals was performed after at least 120 days of treatment. Concentrations of IR5878 in the diet were reduced to 0, 225, 900 and 3600 ppm in order to achieve a constant intake in terms of mg/kg/day and avoid over dosage during lactation of F0 and F1 dams and the rearing of freshly weaned pups.

The overall NOAEL in adult animals (F0 and F1 generation) was established at 1400/900 ppm, based on increased liver and kidney weights at 5600 ppm (F0 males only) and the existence of histopathological changes in liver and kidneys at 5600/3600 ppm in both generations and sexes. The NOAEL for reproductive effects was established at 5600/3600 ppm based on the absence of adverse reproductive effect in both generations. The NOAEL pup development was established at 5600/3600 ppm, on the basis of absence of any developmental effects in both pup generations. The NOAEL pup behaviour was established at 1400/900 ppm, based on reduced locomotor activity in F1 male pups at 5600/3600 ppm.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

400 mg/kg bw/day

Study duration:

chronic

Species:

rat

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

The high food spillage due to the powder diet determined a test item intake unrealistically high. Therefore IR5878 intake was calculated with the mean values obtained in another concurrent 2-generation study performed with same rat strain, and same kind of diet, but pelleted (see point IIA 05.05.01/01). 5600/3600 ppm was determined as the NOAEL for reproductive effects which was a mean intake of 400 mg/kg/day.

Effects on developmental toxicity

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available