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Toxicological information

Toxicity to reproduction

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Administrative data

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

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2003
Report date:
2003

Materials and methods

Test guidelineopen allclose all
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)

Test material

Constituent 1
Test material form:
solid: particulate/powder
Specific details on test material used for the study:
Test material:
IR5878
Batch number: G 009/02
Purity: 98.56 ± 0.19 %

Test animals

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.

Administration / exposure

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.
Doses / concentrationsopen allclose all
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

Examinations

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.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

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 / performance (P0)

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

Details on results (P0)

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.

Effect levels (P0)

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

Results: P1 (second parental generation)

General toxicity (P1)

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 / performance (P1)

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

Results: F1 generation

General toxicity (F1)

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.

Effect levels (F1)

Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
>= 1 400 ppm
Based on:
act. ingr.
Sex:
male
Basis for effect level:
clinical signs

Overall reproductive toxicity

Key result
Reproductive effects observed:
no
Lowest effective dose / conc.:
5 600 ppm (nominal)
Treatment related:
yes

Any other information on results incl. tables

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.

Applicant's summary and conclusion

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.