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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
two-generation reproductive toxicity
Remarks:
based on test type (migrated information)
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
year of publication: 2007
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Acceptable, well-documented publication/study report which meets basic scientific principles

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
2007
Report Date:
2007

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.3800 (Reproduction and Fertility Effects)
Deviations:
no
GLP compliance:
not specified
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): di-2-ethylhexyl terephthalate (DEHT)
- Physical state:liquid
- Analytical purity: > 97%
- Impurities (identity and concentrations): 1.99% 2-ethylhexyl methyl terephthalate
- Stability under test conditions: confirmed (stability, homogeneity and verification of DEHT concentration in feed was confirmed by HPLC/UVD)
- Storage condition of test material: room temperature

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Strain details: Crl:CD(SD)IGS BR rats
- Source: Charles River Laboratories, Raleigh, NC, USA
- Age at arrival: 42 days
- Age at study initiation: 52 days
- Weight at study initiation: data not presented in publication
- Housing:
pre-mating - F0 and F1 parental animals were housed individually in clean, wire-mesh cages
mating: during cohabitation the pairs were left in the home cage of the male animal
after mating: after 14 days or after evidence of mating males were further caged individually in their cages until scheduled necropsy, females were transferred to plastic maternity cages with nesting material (Bed-O Cobs). Damns were housed there until weaning on lactation day (LD) 21

- Diet: fed PMI Nutrition International, Inc., Certified Rodent Lab diet 5002, ad libitum
- Water: municipal water (reverse osmosis treated on-site), ad libitum
- Acclimation period: 10 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): no data
- Humidity (%): no data
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): no data

As the study is performed according to guidelines appropriate conditions are assumed.

Administration / exposure

Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on exposure:
DIET PREPARATION
- Rate of preparation of diet (frequency): every 2 weeks
- Storage temperature of food: room temperature
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: 14 days or until evidence of mating
- Proof of pregnancy: copulatory plug or sperm in vaginal smear referred to as day 0 of pregnancy
- When evidence of mating did not occur within 14 days the female was plaved in a maternity cage with no further opportunity for mating.
- After successful mating each pregnant female was caged (how): in plastic maternity cages with nesting material (Bed-O Cobs) until LD21/PND21
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
stability, homogeneity and verification of DEHT concentration in feed was confirmed by HPLC/UVD
Duration of treatment / exposure:
Parental animals F0: total of 17 to 19 weeks
males: 70 consecutive days before mating, throughout mating until scheduled necropsy (6 to 10 days after weaning of litters);
females: 70 consecutive days before mating, throughout mating, gestation and lactation until scheduled necropsy

Parental animals F1: according to the treatment of F0 test animals
Frequency of treatment:
daily (fed ad libitum)
Details on study schedule:
Parental animals started to receive diet containing the test material when approximately 8 weeks for the F0 generation and at PND22 for the F1 generation. In both cases the animals were treated for 70 days pre-mating thus the F0 animals were approximately 18 weeks old when mating was started. The F1 animals were 13 weeks old when mating was started.
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 3000, 6000, 10000 ppm
Basis:
nominal in diet
No. of animals per sex per dose:
30
Control animals:
yes, plain diet

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule:weekly

BODY WEIGHT: Yes
- Time schedule for examinations:
F0/F1 males: weekly throughout the study and just before necropsy
F0/F1 females: weekly until evidence of copulation, then on GD (gestation day) 0, 4, 7, 11, 14, 17, 20 and on LD 1, 4, 7, 14, and 21

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
F0/F1 males: weekly throughout the study, except during mating
F0/F1 females: weekly except during mating and after evidence of copulation, then on GD (gestation day) 0, 4, 7, 11, 14, 17, 20 and on LD 1, 4, 7, 14, and 21
Oestrous cyclicity (parental animals):
Starting 21 days before mating until the day of verified mating vaginal smears were prepared daily to determine estrous cyclicity. Vaginal smears were carried out on females also on the day of their euthanasia to determine the stage of estrus.
Sperm parameters (parental animals):
Parameters examined in all male parental generations:
testis weight (right and left), epididymis weight (right and left), cauda epididymis weight (right and left) sperm count in testes, sperm count in epididymides, sperm motility, sperm morphology
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 8 pups/litter (equal number/sex/litter as nearly as possible); excess pups were killed

PARAMETERS EXAMINED
The following parameters were examined in F1 / F2 offspring:
All pups were identified and sexed at PND0. Survival and signs of toxicity were examined twice daily, Body weight was assessed on PND 1, 4, 7, 14, and 21. The following parameters were noted: number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, detailed physical examination
In F1 pups selected as next generation balnopreputial separation and vaginal perforation were used to assess sexual maturation.

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities (using different methods when pups died either inbetween PND0 and 4 or after PND4)
Postmortem examinations (parental animals):
SACRIFICE
F0/F1 parental animals were sacrificed 6 to 10 days after weaning of litters. Female animals that experienced total litter loss were euthanized within 24 hours.
F1 weanlings not selected for the next generation and F2 pups were euthanized on PND 21.

GROSS NECROPSY
- Gross necropsy was performed on all parental animals and weanlings, selected organs were weighed.

HISTOPATHOLOGY / ORGAN WEIGHTS
Selective histopathologic examination was carried out on 10 parental animals per gender in the control and high dose groups. In addition, uteri and vaginas from all F0, and F1, adult female animals in the control and high dose groups were examined histopathologically.
Moreover, uteri and vaginas from all F0 and F1 adult female animals in the low- and mid-dose groups with uterine weights > 1 g were also examined.

The following organs were weighed after termination:
brain, liver, kidneys, splee,, seminal vesicles/coagulating gland, prostate, testis (right and left), epididymis (right and left), cauda epididymis (right and left), uterus, ovaries, thymus gland, adrenal glands, pituitary gland
No detailed overview of tissues prepared for microscopic examination is given in the publication.
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring not selected as parental animals were sacrificed on PND4.
- F2 offspring were sacrificed on PND21.
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination).

GROSS NECROPSY
- Gross necropsy was performed, but no details are given within the publication.

HISTOPATHOLOGY / ORGAN WEIGTHS
No details are given in the publication.
Statistics:
The study was performed using two-tailed analysis with a minimum significance level of 5%.
The following statistical analyses were carried out:
- for parental mating and fertility indices: chi square test with Yates' correction factor
- for body weights (adult and pup from PND 4 to 21), feed consumption, organ weights, sperm numbers, precoital intervals, live litter size, aquistion of developmental landmarks: one-way analysis of variance (ANOVA) with Dunnett's test
- for sperm motility, sperm morphology, pup gender ratio, postnatal survival: Kruskal-Wallis test with Mann-Whitney U test
- for histopathological data: Fisher's exact test
- for offspring weights before standardisation on PND4: analysis of covariance and Student's t-test
Reproductive indices:
To assess reproductive parameters the following indices were given:
- estrous cycle length (days)
- precoital interval (days)
- mating index (%)
- fertility index (%)
- gestation length (days)

No details on calculation of the indices were given in the publication.
Offspring viability indices:
The following offspring indices were given:
- total pups/litter (n)
- live pups/litter (n; mean live litter size)
- gender ratios (% per litter)
- postnatal pup survival (% per litter) on PND 0, PND 0 to1, PND 1 to 4, PND 0 to4, PND 4 to 21

No details on calculation of the indices were given in the publication.

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Other effects:
no effects observed

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)

Mortality:
F0:
- 10000 ppm: 3 female rats 2 to 8 days after weaning of F1 pups were found dead or were sacrificed in extremis
F1:
- 10000 ppm: 7 female rats 2 to 8 days after weaning of F2 pups were found dead or were sacrificed in extremis

Single male deaths in F0 control and mid dose group as well as in the F1 high dose group were not attributed to substance treatment by the authors. All other F0/F1 animals survived to scheduled necropsy.

Body Weights:
F0:
- 10000 ppm:
males: reduced mean weekly bw gain (15-25%) during weeks 3 to 4, 4 to 5, and 6 to 7 resulted in a slightly reduced bw at termination (5%) in this group
females: reduced bw gain during gestation (12%), resulting in reduced bw during gestation and throughout lactation
- 3000 and 6000 ppm: No test article-related effects on mean weekly body weights or other parameters in correlation to bw of male and female animals were seen.
F1:
- 10000 ppm:
males: reduced mean body weight, along with lower mean birth weights and decreased growth before weaning from the F0 maternal animals, resulted in reduced mean body weights until scheduled necropsy (-13% at termination)
females: resulting from reduced mean bw during the pre-weaning period, pre-mating bw were decreased relative to the control (statistically significant); comparable to F0 reduced bw gain during gestation, resulting in reduced bw during gestation and throughout lactation, final body weight - 6%
- 6000 ppm:
males reduced mean body weight, along with lower mean birth weights and decreased growth before weaning from the F0 maternal animals, resulted in reduced mean body weights until scheduled necropsy (-6% at termination)
females: resulting from reduced mean bw during the pre-weaning period, pre-mating bw were decreased relative to the control (not statistically significant); reduced mean bw during GD11 to 20 and LD1 to 14, final body weight - 6,5%
- 3000 ppm: No test article-related effects on mean weekly body weights or other parameters in correlation to bw of male and female animals were seen.

Food Consumption:
The authors calculated the mean compound consumption (for details see table in 'Any other information').
F0:
- 1000 ppm:
females: reduced throughout gestation and lactation (statistically significant; correlating with reduced bw)
F1:
- 10000 ppm
males: slightly reduced throughout generation (correlating with reduced bw)
females: reduced throughout gestation and lactation (statistically significant; correlating with reduced bw)
- 6000 ppm:
males: slightly reduced during the first week after weaning (correlating with reduced bw)
females: slightly reduced from PND7 to 14 (correlating with reduced bw)

There were no other treatment related changes in food consumption (g/kg/day) in the remaining male and female treatment groups.

Organ Weights:
F0:
- 6000 &10000 ppm: females: relative liver wt increased
F1:
- 6000 &10000 ppm: females: relative liver wt increased

Several statistically significant decreases were observed in mean absolute organ weights in the F0 and F1 adults (e.g. spleen, kidneys, uterus, testes). In most cases, these changes disappeared when compared relative to the body weight. This suggests the differences were due to the decreased body weights. Moreover there were no correlative findings in the respective organs in macroscopic and microscopic examinations. Uterine weights in controls were unusually high due to the fact that a lot of the animals were in estrous or proestrous on the day of necropsy. Similarities between the uterine weights from F1 treatment groups with the F0 control further suggests that the effect is not substance related.


Histopathology: There were no test substance-related microscopic lesions in any of the tissues examined for the adult F0 and F1 rats.

Effect levels (P0)

open allclose all
Dose descriptor:
NOAEL
Remarks:
reproduction
Effect level:
10 000 ppm (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no reproductive toxicity was observed up to the highest dose level and under the conditions of this study
Remarks on result:
other: corresponding to 149-205/198-450 mg 2-EH/kg bw/day in F0 males/females or 184-298/232-516 mg 2-EH/kg bw/day in F1 males/females
Dose descriptor:
NOAEL
Remarks:
parental toxicity
Effect level:
3 000 ppm (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
mortality
body weight and weight gain
organ weights and organ / body weight ratios
Remarks on result:
other: high dose: mortality F0&F1 maternal animals, decreased mean body weights in m/f animals of F0 & F1, increased relative liver weights (F0 & F1 females); mid dose: slight decrease of mean bw in F1, increase in relative liver weights (F0 & F1 females)

Results: P1 (second parental generation)

General toxicity (P1)

Clinical signs:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Other effects:
no effects observed

Reproductive function / performance (P1)

Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed

Details on results (P1)

For details on results see field "Details on results (P0)"

Effect levels (P1)

open allclose all
Dose descriptor:
NOAEL
Remarks:
reproduction
Effect level:
10 000 ppm (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no reproductive toxicity was observed up to the highest dose tested and under the conditions of this study
Remarks on result:
other: corresponding to 14 9-205/198-450 mg 2-EH/kg bw/day in F0 males/females or 184-298/232-516 mg 2-EH/kg bw/day in F1 males/females
Dose descriptor:
NOAEL
Remarks:
parental toxicity
Effect level:
3 000 ppm (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
mortality
body weight and weight gain
organ weights and organ / body weight ratios
Remarks on result:
other: high dose: mortality F0&F1 maternal animals, decreased mean body weights in m/f animals of F0 & F1, increased relative liver weights (F0 & F1 females); mid dose: slight decrease of mean bw in F1, increase in relative liver weights (F0 & F1 females)

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:
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
no effects observed
Histopathological findings:
not specified

Details on results (F1)

Body Weights: (litter effect was accounted for: i.e. number of pups born per litter affetcs the mean pup weight: in litters with larger number of animals the mean pup bw is lower and vice verca; litter sizes can be found in detail in 'Any other information')
F1 (pups)
- 6000 & 10000 ppm: reduced postnatal pup body weights in early postnatal phases and later on at PND14 to 21 and pup body weight gains (decreases in feed consumption in the female animals from this group during gestation and lactation may have contributed to the early effects, reductions in pup body weight gain later in lactation may have been due to direct consumption of the treated feed or taste aversion to the same)

F2:
- 10000 ppm: reduced postnatal pup body weights in early postnatal phases and later on at PND14 to 21
- 6000 ppm:reduced postnatal pup body weights only at PND14 to 21

No other relevant differences in mean body weight or body weight gains were observed among any group.

Food Consumption: Not Applicable

Developmental Landmarks:
F1 (weanlings):
- 10000 ppm: approx. 2 days delay in age of acquisition of balanopreputial separation, most probably due to the reduced body weight
- in female animals mean age of vaginal patency was similar between control group and all treatment groups.
The body weights on the day of attainment were decreased (significant statistically) at all dose levels reflecting either the slightly younger age of the animals (3000 ppm group) or the reduced body weights (6000 and 10000 ppm groups).

Organ Weights: all changes were noted on PND21
F1 (weanlings):
- 10000 ppm:
males: decreased relative spleen weight (13%), increased mean relative brain weight (25%)
females: increased mean relative brain weight (25%)
- 6000 ppm:
females: increased mean relative brain weight (12%)

F2:
- 10000 ppm:
males: decreased relative spleen weight (8%), increased mean relative brain weight (23-25%)
females: decreased relative spleen weight (11%), decreased relative thymus weight (12%), increased mean relative brain weight (23-25%)

All other organ weight changes noted disappeared when when adjustment to decreasd body weight was performed.

Necropsy/Histopathology: No relevant macroscopic findings were noted in F1 and F2 pups. No details of microscopic examinations were given in the publication.

Reproductive parameters: The parameters investigated (estrous cycle length, time to mating (precoital interval), mating and fertility indices, gestation length) were unaffected during the F0 and F1 generations.

Litter parameters: Number of pups born per litter, mean live litter sizes, gender ratio, and postnatal survival were unaffected by the test substance in the F0 and F1 generation.

Spermatogenic Endpoints: For none of the measured parameters in relation to sperm quality a toxicologically significant difference was noted in the F0 and F1 generations.

Effect levels (F1)

Dose descriptor:
NOAEL
Remarks:
developmental toxicity
Generation:
F1
Effect level:
3 000 ppm (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
Remarks on result:
other: high and mid dose group: reduced m/f pup weights on PND14 to21 in F1 and F2 generation

Results: F2 generation

General toxicity (F2)

Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Sexual maturation:
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
no effects observed
Histopathological findings:
not specified

Details on results (F2)

For details on results please see "Details on results (F1)"

Effect levels (F2)

Dose descriptor:
NOAEL
Remarks:
developmental toxicity
Generation:
F2
Effect level:
3 000 ppm (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
Remarks on result:
other:
Remarks:
high and mid dose group: reduced m/f pup weights on PND14 to21 in F1 and F2 generation

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

Mean calculated compound intake [mg/kg/day]

   Males     Females         
 Nominal dietary level  before breeding  after breeding  before breeding  gestation  lactation  after weaning
 F0                  
 3000 ppm  182 133  223  184 478  207 
 6000 ppm  367 265  449  372  940  419 
 100000 ppm  614 447  783  595  1349  745 
 F1                  
 3000 ppm  256 159  275  206  516  227 
 6000 ppm  523 320  573  423  1036  486 
 10000 ppm  893 552  1021  697  1549  868 

Live litter size [n]

               Dose group
   control  3000 ppm 6000 ppm  10000 ppm 
 F1  13.0 +/- 2.72   14.4 +/- 3.34   14.3 +/- 1.54   13.3 +/- 2.88
 F2   13.9 +/- 3.18   14.0 +/- 3.16   14.2 +/- 2.02   13.7 +/- 2.83

Applicant's summary and conclusion

Conclusions:
In a 2-generation guideline study 30 rats/sex/group/generation were exposed to di (2-ethylhexyl) terephthalate ad libitum in the diet at dose concentrations of 0, 3000, 6000, and 10000 ppm. No adverse effects were found on reproductive parameters such as estrous cyclicity, gonadal functions, spermatogenic endpoints (motility, morphology, counts), mating behaviour and performance, conception, gestation and parturition, and fertility in general. No adverse effects on the reproductive organs were noted.
Effects observed included unspecific systemic toxicity. In the high dose group mortality in F0 and F1 maternal animals was observed, a decrease of mean body weights in m/f animals of F0 and F1 generation was noted (various time points during the study) and an increase in relative liver weights (F0 and F1 females) found. In the mid dose group effects observed included a slight decrease of mean body weights in F1 generation (various time points during the study) and an increase in relative liver weights (F0 and F1 females). the increases in liver weights were not accompanied by any macroscopic or microscopic findings. This holds also true for all other organs examined during necropsy and histopatholgical evaluation. In the high and mid dose groups reduced postnatal pup weights were observed for both sexes in the F1 and F2 generation.
As there were no effects on any of the measured parameters for effects on reproduction, the no-observed-adverse-effect level (NOAEL) for reproductive toxicity was considered to be 10000 ppm.
Based on the described effects of general systemic toxicity at the mid and high dose group, the NOAEL for parental toxicity was considered to be 3000 ppm.
The effects observed in the neonates (lower body and organ weights, both effects in the presence of maternal toxicity) lead to the conclusion that the NOAEL for developmental toxicity can be established at 3000 ppm.
Executive summary:

Thirty Crl:CD®(SD)IGS BR rats/sex/group/generation were exposed to di (2-ethylhexyl) terephthalate in the diet at dose concentrations of 0, 3000, 6000, and 10000 ppm in this two generation reproduction toxicity study according to OECD guideline 416. Treatment of parental animals in the F0 generation started at 52 days of age and at postnatal day 22 (PND22) for the F1 generation. Animals received the diets for 70 consecutive days prior to mating, through the mating period (maximum of 14 days), during gestation and lactation until their scheduled necropsy (F0/F1 parental animals: 6 -8 days after weaning, F1 pups not selected for further generation and F2 pups at PND21).

In guideline conform intervals cage side and detailed clinical observations, measurements of body weight and food consumption were performed. Various reproductive and developmental indices were assessed ( estrous cyclicity, gonadal functions, spermatogenic endpoints (motility, morphology, counts), mating behaviour and performance, conception, gestation and parturition, and fertility in general; total pup number/litter, mean live litter size, gender ratios, postnatal pup survival, developmental landmarks such as balanopreputial separation and vaginal patency).

Parturition was allowed naturally as well as rearing of pups until weaning. On PND4 thirty F1 pups/sex/group were selected to constitute the F1 generation. 

Gross necropsies were performed on all animals. Histopathological examinations on various organs and tissues were performed on parental animals whereas for pups only organ weights were determined.     

Substance treatment had no adverse toxicological effect on any of the measured reproductive parameters for the F0 and F1 generations. Therefore the no-observed-adverse-effect level (NOAEL) for reproductive toxicity was considered to be 10000 ppm.

In the high dose group deaths of 3 damns in the F1 generation and 7 damns in the F2 generation were attributed to substance treatment, as the deaths occurred 2 to 8 days after weaning - a period in which the damns maintain their body weights while still consuming large amounts of feed and thus resulting in high dose levels in the body (i.e. F0 = 745 mg/kg x d; F1 = 868 mg/kg x d). Single deaths in male animals were not treatment related. In high dose female animals of the F0 and F1 generation mean maternal body weights were reduced during gestation and lactation. Food consumption was also reduced in these dose groups during this time of exposure. In high dose group males of the F0 generation transient reductions of mean body weights occurred during various time points of the study. In the F1 generation males of the high and mid dose group showed reduced body weights starting from birth throughout the study. This was also true for females of the F1 in the mid dose group. Food consumption was reduced in males of the F1 generation in the high dose group throughout the study, whereas in the mid dose group this reduction was limited to the first week after weaning. In the high dose group increases in relative liver weights were observed in F0 and F1 females. This effect was not accompanied by any gross or microscopic findings and thus is not accounted for as adverse. Taken together these results indicate that a NOAEL for parental toxicity can be established at 3000 ppm.

In the high and mid dose group mean F1 and F2 male and female offspring weights were reduced on PND14 to 21. In the high dose group also reduced pup weights were observed at earlier time points (PND 1 and 7). In the high dose group in both sexes of F1 and F2 pups increased relative brain weights were found. In males of both generations decreased relative spleen weights were also observed. In female animals of the F2 generation reduced relative spleen and thymus weights were noted. In the mid dose group increased relative brain weights were found in females of the F1 generation. Based on these results, the NOAEL for developmental toxicity was considered to be 3000 ppm (Faber at a., 2007).