Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records
Reference
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
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
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.
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.
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
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.
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: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed
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.
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)
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: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed
For details on results see field "Details on results (P0)"
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)
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
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.
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
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
For details on results please see "Details on results (F1)"
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
Reproductive effects observed:
not specified

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
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).

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
149 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
Reliable guideline study (Klimisch score 2)
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

There is no valid fertility study available for 2 -ethylhexan-1 -ol.

However read-across was performed to di (2 -ethylhexyl) terephthalate (DEHT) to cover the endpoint’s requirements.

This was considered adequate as

-         DEHT is metabolised immediately and entirely to 2-EH and terephthalic acid. The submission substance is thus available in the body after DEHT application (for further details please see section on toxicokinetics and paragraphs below).

-         The highest dose tested in the two generation study with DEHT is in the range of the maximum tolerable dose (MTD) for 2-EH as required by the testing guideline.

For estimating the maximum possible dose of 2-EH in the two generation study with DEHT the following assumptions were made:

-Only 50% absorption of DEHT: In the gavage study by Barber et al. (1994) approximately 65 mg of 100 mg DEHT administered were absorbed (for details please cf. to section on toxicokinetics). Therefore the approach taken is conservative as the maximum height of 2-EH dose reached after DEHT administration is critical for assessing the validity of the study for read-across.

-Complete metabolism of DEHT to 2-EH (1 mole DEHT leads to 2 mole of 2-EH; for details please cf. to section on toxicokinetics).

The tested doses of DEHT correspond to the following maximum doses of 2-EH

-F0 m/f: 10000 ppm = 447-614/595-1349 mg DEHT/kg/d,

corresponding to 149-205/198-450 mg 2-EH/kg/d

-F1 m/f: 10000 ppm = 552-893/697-1549 mg DEHT/kg/d,

corresponding to 184-298/232-516 mg 2-EH/kg/d

-         Observed effects (decrease of body weight) are comparable at approximately equivalent 2 -EH doses in between the two generation study performed with DEHT and the 90-day toxicity study in rats using 2-EH as test material (Astill et al., 1996).

In this two generation reproduction toxicity study according to OECD guideline 416 thirty Crl:CD®(SD)IGS BR rats/sex/group/generation were exposed to di (2-ethylhexyl) terephthalate (DEHT) in the diet at dose concentrations of 0, 3000, 6000, and 10000 ppm. Treatment of parental animals in the F0 generation started at 52 days of age and 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 wells 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 (F0 m/f: 447 -614/595/1349; F1 m/f: 552 -893/697-1549 mg DEHT/kg x d).

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 DEHT/kg x d; F1 = 868 mg DEHT/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 (133 to 516 mg DEHT/kg x d).

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 et al., 2007).

Furthermore two non guideline in vitro and in vivo studies applying 2-EH are available.

- When 2-EH was administered to cells of mouse Leydig tumour cell line, MA-10, in concentrations up to 1 mM for at least 24 hours the test material did not reveal any cytotoxic effects nor did 2-EH reduce steroidogenic potential of the cells (no reduction of progesterone production or gene expression of key steroidogenic enzymes; Piche et al., 2012, cf. IUCLID section 7.9.3).

- The effects of 2-EH (and also of DEHP, MEHP, and 3 metabolites of MEHP; all test substances were administered at 2.7 mmol/kg bw/day for 5 consecutive days) were examined in vivo and in vitro by Sjöberg et al., 1986. No testicular damage was seen in rats that were dosed with 2-EH, i.e. the number of degenerated cells in the seminiferous tubules was not increased, whereas this was the case with MEHP. This was supported by the observation that MEHP in the range 1–200 µM increased significantly the detachment of germ cells in primary rat testicular cell cultures. 2-EH had no effect at 200µM. It was concluded that MEHP, but not 2-EH was responsible for testicular damage observed after dosing DEHP (cf. IUCLID section 7.9.3 ).

Taken the results of the two generation study with DEHT together with studies for 2-EH which demonstrate the lack of toxicity to reproduction, i.e.

- no adverse effect on any gestational parameter in prenatal developmental studies with various routes of exposure (NTP 1991- oral; Tyl et al, 1992 –dermal; Nelson et al., 1989 –inhalation; cf. IUCLID section 7.8.2),

- no effect on testes and ovaries of rats and mice in 90-d oral gavage studies (Astill et al., 1996; cf. IUCLID section 7.5.1),

- no anti-androgenic activity in vitro (Piche et al., 2012), or

- no degeneration of testes (in vivo) and sertoli cells (in vivo and in vitro)

thus give the overall evidence that 2 -EH is not triggering adverse effects on reproduction.

 

REMARK:

2-ethylhexanol was tested in a reproduction toxicity screening test under GLP at 1525 mg/kg bw/day in time-pregnant female mice (50/group). The animals were dosed by oral gavage during gestation days 7 through 14. This dose caused severe maternal toxicity and extensive mortality (substance-related mortality 34%). This, and also other shortcomings of the study (e.g. only female animals, only one dose level tested, dose level was too high, treatment period too short), invalidates the study, because the observed effects on reproduction parameters (increased resorptions and nonviable pups; reduced number of pups/litter, reduced pup weight; reduced pup weight gain) could be related to the maternal toxicity. This study is therefore regarded to be not reliable (Hazleton, 1983).


Short description of key information:
Taken the results of a two generation study with DEHT (precursor of 2-EH) together with various studies for 2-EH which demonstrate the lack of toxicity to reproduction (e.g no adverse effect on any gestational parameter in prenatal developmental studies, no effect on testes and ovaries of rats and mice in 90-d oral gavage studies, no anti-androgenic activity in vitro or degeneration of testes (in vivo) and sertoli cells (in vivo and in vitro)) are supporting the notion that 2-EH is not a reproductive toxicant.

Justification for selection of Effect on fertility via oral route:
The results from a two generation reproduction toxicity study according to OECD guideline 416 using di(2 -ethylhexyl) terephthalat were considered adequate to cover this endpoint's requirements as the submission substance is an immediate metabolite of the test material used (for further details please see section on toxicokinetics). The NOAEL identified in the study was 10000 ppm in diet, this corresponds to 447-614/595-1349 mg DEHT/kg bw/day in F0 male and female animals or 552-893/697-1549 mg DEHT/kg bw/day for F1 male and female animals.This approximates to F0 m/f = 149-205/198-450 mg 2-EH/kg bw/day or F1 m/f = 184-298/232-516 mg 2-EH/kg bw/day.

Effects on developmental toxicity

Description of key information
No signs of maternal or developmental toxicity were seen in mice at 191 mg/kg bw/d (oral); the NOAEL was 191 mg/kg bw/day. In a gavage study in rats developmental toxicity as well as teratogenic effects were observed at the highest dose group tested (1300 mg/kg bw/d), but only in the presence of sever maternal toxicity.
No maternal or developmental toxicity or teratogenicity was seen after inhalation exposure of rats at 850 mg/m³ within gestation days 0-19 for 7 hrs/day.
Following dermal application during gestational days 6-15, local skin irritation was seen at 850 mg/kg bw/day; slight maternal toxicity at 2520 mg/kg bw/d but no developmental toxicity or teratogenicity was seen up to the highest dose tested (2520 mg/kg bw/d).
Overall:
After administration of 2-EH only one study showed developmental toxic and teratogenic effects. However both effects were only observed in the presence of severe maternal toxicity including mortality.
Link to relevant study records

Referenceopen allclose all

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
Year of publication: 1991
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Comparable to guideline study
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
mouse
Strain:
CD-1
Details on test animals and environmental conditions:
- CD-1 Swiss mice
TEST ANIMALS
- Source: Charles River Laboratories Inc., Raleigh, NC, USA
- Weight at study initiation: range 23.52-31.59 g
- Housing: individually in solid-bottom polycarbonate cage swith stainless steel wire lids
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 7 days


ENVIRONMENTAL CONDITIONS
- Temperature: 72°F
- Humidity (%): 48
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:
oral: feed
Vehicle:
other: microencapsulation
Details on exposure:
DIET PREPARATION
- Rate of preparation of diet (frequency): fed was prepared once. Fresh supplies of dosed feed were obtained from refrigerated stock every 3 days.
- Mixing appropriate amounts with (Type of food): Ground Purina Certified Rodnet Chow
- Storage temperature of food: refrigerated


Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Concentration of 2-EH in the feed was analyzed by gas chromatography (GC) prior to use.
Details on mating procedure:
- Impregnation procedure: purchased timed pregnant
Duration of treatment / exposure:
Gestational days 0 to 17
Frequency of treatment:
7/week
Duration of test:
17 days
Remarks:
Doses / Concentrations:
0, 17, 59, 191 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
28
Control animals:
yes, plain diet
Details on study design:
Microencapsulated 2-EH (0%, 0.009%, 0.03%, or 0.09% in feed) was provided on gestational days (gd) 0 to 17 ad libitum to timed-mated CD-1® mice
(28/group).
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily
- Cage side observations checked in table [Appendis I-11] were included.


BODY WEIGHT: Yes
- Time schedule for examinations: gestational day 0, 3, 6. 9. 12, 15, 17


FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes


POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 17
- Organs examined: liver and uterus


Ovaries and uterine content:
At sacrifice (gd 17), the number of ovarian corpora lutea and uterine implantation sites, including resorptions, and dead or live fetuses, were recorded.
Fetal examinations:
Live and dead fetuses were weighed. Live fetuses were sexed and examined for external, visceral and skeletal malformations and variations.
Statistics:
General Linear Models (GLM) procedures were applied for the analysis of variance (ANOVA) of maternal and fetal parameters. Bartlett's test for homogeneity of variance was performed an all data to be analyzed by ANOVA. When ANOVA revealed a significant (p<0.05) dose effect, Dunnett’s Multiple Comparison Test was used to compare each of the treated groups with the control groups. Other analyses comprised chi square test and Fisher’s exact probability test.
Indices:
none
Historical control data:
not required
Details on maternal toxic effects:
Maternal toxic effects:no effects

Details on maternal toxic effects:
No dams died, delivered early or were removed from study. Pregnancy rate was high (93-96%) and equivalent across all groups. One litter at 0%
was fully resorbed; all other pregnant animals had live litters at scheduled necropsy. The numbers of live litters evaluated were 27 at 0.009 and
0.03% and 26 at 0 and 0.09% 2-EH.
There was no treatment-related maternal toxicity observed in this study. Maternal body weights, weight gains (absolute or corrected for gravid
uterine weight), gravid uterine weight and liver weight (absolute or relative to body weight) were unaffected. Food consumption (g/kg/day and g/day) was significantly increased for gd 0-3 at 0.09% and unaffected for all other time points evaluated. The calculated consumption of 2-EH, based on gestational food consumption was 0 (0 mmol/kg), 17 (0.13 mmol/kg), 59 (0.46 mmol/kg) and 191 mg/kg/day (1.49 mmol/kg), for the 0, 0.009, 0.03 and 0.090% groups, respectively.

Dose descriptor:
NOAEL
Effect level:
191 mg/kg bw/day (actual dose received)
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOAEL
Effect level:
191 mg/kg bw/day (actual dose received)
Basis for effect level:
other: developmental toxicity
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
There were no effects of exposure to dietary 2-EH on any gestational parameters. The number of corpora lutea, uterine implantation sites (live, dead, resorbed), pre- and postimplantation loss, sex ratio (%, males) and live fetal body weight per litter (all fetuses or separately by sex) were all equivalent across all groups. There were also no treatment-related changes in the incidence of individual, external, visceral, skeletal or total malformations or variations.

Dose descriptor:
NOAEL
Effect level:
191 mg/kg bw/day (actual dose received)
Basis for effect level:
other: teratogenicity
Abnormalities:
not specified
Developmental effects observed:
not specified

No dams died, delivered early or were removed from study. Pregnancy rate was high (93-96%) and equivalent across all groups. One litter at 0% was fully resorbed; all other pregnant animals had live litters at scheduled necropsy. The numbers of live litters evaluated were 27 at 0.009 and 0.03% and 26 at 0 and 0.09% 2-EH. There was no treatment-related maternal toxicity observed in this study. Maternal body weights, weight gains (absolute or corrected for gravid uterine weight), gravid uterine weight and liver weight (absolute or relative to body weight) were unaffected. Food consumption (g/kg/day and g/day) was significantly increased for gestational das 0-3 at 0.09% and unaffected for all other time points evaluated. The calculated consumption of 2-EH, based on gestational food consumption was 0 (0 mmol/kg), 17 (0.13 mmol/kg), 59 (0.46 mmol/kg) and 191 mg/kg/day (1.49 mmol/kg), for the 0, 0.009, 0.03 and 0.090% groups, respectively.

There were no effects of exposure to dietary 2-EH on any gestational parameter. The number of corpora lutea, uterine implantation sites (live, dead, resorbed), pre- and postimplantation loss, sex ratio (%, males) and live fetal body weight per litter (all fetuses or separately by sex) were all equivalent across all groups. There were also no treatment-related changes in the incidence of individual, external, visceral, skeletal or total malformations or variations.

Conclusions:
No maternal or developmental toxicity was observed in a mouse oral feed study at (equivalent to OECD TG 414, dosing during gestation day 0-17) at any dose up to and including 191 mg/kg bw/day, the highest tested dose.
At equimolar doses, DEHP and and MEHP caused both maternal and developmental toxicity. It was therefore concluded that 2-EH does not play a in DEHP- or MEHP toxicity.
Executive summary:

2-EH was examined in a mouse feed study for its potential for developmental toxicity equivalent to OECD TG 414 and under GLP conditions. Timed pregnant female CD-1 Swiss mice (28 animals/group, body weight range 32.5 to 31.6 g) received 2-EH in the diet at nominal concentrations of 0, 0.009, 0.03, and 0.09% during gestation days 0-17. The animals were housed singly and observations for clinical signs were made daily. Body weights were recorded on gestational day 0, 3, 6, 9, 12, 15, 17. Food consumption and test compound intake were calculated individually. Test substance purity and concentration in thediets wasverified using gas chromatography. Test substance purity was >99%. Concentration in the diets was within 99-108% of the nominal concentration.

 

Maternal effects:

Food intake and hence dose levels were higher than expected. Average intakes were 0, 17, 59, and 191 mg/kg bw/day, respectively. No dams died, delivered early or were removed from study. Pregnancy rates were high (93-96%) and equivalent across all groups. One litter at 0% was fully resorbed; all other pregnant animals had live litters at scheduled necropsy. The numbers of live litters evaluated were 27 at 0.009 and 0.03% and 26 at 0 and 0.09% 2-EH.

There was no treatment-related maternal toxicity observed in this study. Maternal body weights, weight gains (absolute or corrected for gravid uterine weight), gravid uterine weight and liver weight (absolute or relative to body weight) were unaffected. Food consumption (g/kg/day and g/day) was significantly increased for GD 0-3 at 0.09% and unaffected for all other time points evaluated. The calculated consumption of 2-EH, based on gestational food consumption was 0 (0mmol/kg), 17 (0.13mmol/kg), 59 (0.46mmol/kg) and 191 mg/kg/day (1.49mmol/kg), for the 0, 0.009, 0.03 and 0.090% groups, respectively.

 

Fetal effects

There were no effects of exposure to dietary 2-EH on any gestational parameters.The number of corpora lutea, uterine implantation sites (live, dead, resorbed), pre- and postimplantation loss, sex ratio and live fetal body weight per litter (all fetuses or separately by sex) were all equivalent across all groups. There were also no treatment-related changes in the incidence of individual, external, visceral, skeletal or total malformations or variations.

 

In conclusion, there were no maternal or developmental toxic effects of 2-EH dietary exposure throughout gestation at any concentration tested, in contrast to the qualitatively similar maternal and developmental toxicity previously reported for DEHP (Tyl et al., 1988) and MEHP (NTP, 1990) at approximately equimolar doses administered under comparable experimental conditions. The present study therefore indicates that 2-EH plays essentially no role in the expression of DEHP-induced maternal and developmental toxicity. The NOAEL for maternal toxicity and for developmental toxicity and teratogenicity was therefore 191 mg/kg bw/day, the highest dose level tested.

 

This study was conducted equivalent to OECD TG 414 and under GLP conditions, and it is considered to be valid without restriction (Tyl et al., 1991).

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
Year of publication 1989/1990
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Comparable to guideline study with acceptable restrictions. Restriction: low number of dams.
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
yes
Remarks:
low number of animals: 15 females instead of 25
GLP compliance:
not specified
Limit test:
yes
Species:
rat
Strain:
Sprague-Dawley
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratories, Portage, MI, USA
- Age at study initiation: no data
- Weight at study initiation: 200-300 g at beginning of pregnancy
- Fasting period before study: no
- Housing: singly, in stainless steel mesh wire cages during gestation and exposure
- Diet: ad libitum (not during exposure)
- Water: ad libitum (not during exposure)
- Acclimation period: 1-2 weeks


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 24+/- 2
- Humidity (%): 50+/-10
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:
inhalation
Type of inhalation exposure (if applicable):
whole body
Remarks:
air
Vehicle:
other: air
Details on exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: 0.5 m³ whole body exposure chamber (hinners-type)
- Method of holding animals in test chamber: animals were caged
- Source and rate of air: heated compressed air
- Method of conditioning air: heating
- System of generating particulates/aerosols: a constant flow of reagent grade 2-EH was mixed with a metered volume of heated air (<80F°; i.e.<27°C). The vapor-air mixture was introduced into the mainstream of teh chamber airflow.
- Temperature, humidity, pressure in air chamber: 77+/-2F° (i.e. 25°C); relative humididty 50+/-15%
- Air flow rate: 0.5 m³/min
- Air change rate: 60/h
- Treatment of exhaust air: no data


TEST ATMOSPHERE
- Brief description of analytical method used:
(1) continous monitoring using a Miran1A infrared analyzer; records were taken every hour. Calibration checks were completed daily before and after the exposures.
(2) Charcoal tube samples were drawn 2 days/week and analyzed by gas chromatography, with partial verification of spiked samples of known concentration.
- Samples taken from breathing zone: yes


Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
- Brief description of analytical method used:
(1) continous monitoring using a Miran1A infrared analyzer; records were taken every hour. Calibration checks were completed daily before and after the exposures.
(2) Charcoal tube samples were drawn 2 days/week and analyzed by gas chromatography, with partial verification of spiked samples of known concentration.
Details on mating procedure:
details not reported
Duration of treatment / exposure:
gestation day 1 - 19
Frequency of treatment:
daily, 7 hr/day
Duration of test:
19 days
Remarks:
Doses / Concentrations:
850 mg/m³
Basis:
analytical conc.
No. of animals per sex per dose:
15 females
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: the highest achievable concentration was used (850 mg/m³) which could be generated as a vapor while maintaining the chamber temperature <26°C. As no maternal toxicity was noted, there was no need to test lower concentrations.
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly


BODY WEIGHT: Yes
- Time schedule for examinations: daily for the first week and weekly thereafter


FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No; data were calculated on a weekly basis


WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No; not applicable
- Time schedule for examinations:


POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organs examined: uterus, ovaries

Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: No data
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
Fetal examinations:
- External examinations: Yes:all per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: Yes: No data
Statistics:
Multivariate analysis of variance (MANOVA) and analysis of variance (ANOVA) were used.
Details on maternal toxic effects:
Maternal toxic effects:no effects

Details on maternal toxic effects:
There was no effect except reduced feed consumption (approx. -10% compared to controls) and reduced body weight gain (-20% vs. controls) during gestation.
Dose descriptor:
NOAEC
Effect level:
ca. 850 mg/m³ air
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOAEC
Effect level:
ca. 850 mg/m³ air
Basis for effect level:
other: developmental toxicity
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
see table below
Dose descriptor:
NOAEC
Effect level:
ca. 850 mg/m³ air
Basis for effect level:
other: teratogenicity
Abnormalities:
not specified
Developmental effects observed:
not specified

    

There were no external, skeletal, or visceral malformations observed in any group.

Conclusions:
No signs of maternal and fetal toxicity were noted in this inhalation study, where the maximum vapour concentration was used which does not lead to formation of aerosol.
Executive summary:
No maternal toxicity or developmental toxicity was noted in a rat inhalation study that was conducted similar to OECD TG 414; the study is valid though less animals (n = 15) than suggested by the test guideline. The rats were exposed during days 0 -19 of gestation to 2 -EH at 850 mg/m³, the maximum achievable concentration without aerosol formation.
There were only minor signs of maternal toxicity, i.e. reduction of feed consumption (p<0.05) and body weight gain during gestation. The reproduction parameters were unchanged. There was no indication of developmental toxicity or teratogenicity, as the incidences of resorptions, the number of fetuses per litter, the sex ration, fetal weight, were all comparable to the control group. Moreover, there were no external, skeletal or visceral malformations observed in any group. 
The authors concluded that due to the low vapor pressure the systemic dose was too low to produce maternal or developmental toxicity. This does not only apply for 2 -EH, but also for other alcohols with chain lengths of 5 or more carbons. Smaller alcohols with chain lengths of 1 to 4 carbons are much more volatile, and produce maternal toxicity, and also developmental toxicity in the presence of maternal toxicity. 
The inhalation NOAEL for maternal toxicity and developmental toxicity and teratogenicity was therefore 850 mg 2 -EH/m³.
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
year of publication: 1992
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Comparable to guideline study
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Fischer 344
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratories, Kinston, NY
- Age at study initiation: 84 (m) and 67 days (f)
- Weight at study initiation: 175-200 g (m) and 130-150 g (f) on arrival
- Fasting period before study: no data
- Housing: pregnant females were housed singly
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 14 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-23
- Humidity (%): 42-65
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12/12


Route of administration:
dermal
Vehicle:
unchanged (no vehicle)
Details on exposure:
TEST SITE
- Area of exposure: clipped dorsal skin, 1.5 x 1.5 inch, i.e. 9.7 cm²
- % coverage: no data
- Type of wrap if used: gauze patch, covered by a polyethylene patch. The application site was occluded with a Lycra-Spandex jacket
with Velcro closures.
- Time intervals for shavings or clipplings: no data


REMOVAL OF TEST SUBSTANCE
- Washing: no; th application site was gently wiped with moist gauze and blotted dry
- Time after start of exposure: 6 hours


TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.3, 1, and 3 ml/kg bw/day
- Concentration (if solution): undiluted
- Constant volume or concentration used: yes; based on body weight on gestational day 6
- For solids, paste formed: n.a.



USE OF RESTRAINERS FOR PREVENTING INGESTION: no
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The purit of the test material was examined by gas chromatography. The test material was dispensed from a 1.0 mL syringe.
Details on mating procedure:
- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1:1
- Length of cohabitation: no data
- Verification of same strain and source of both sexes: yes
- Proof of pregnancy: vaginal plug
Duration of treatment / exposure:
gestation day (GD) 6 through 15
Frequency of treatment:
daily; 6 hours/day
Duration of test:
21 days
Remarks:
Doses / Concentrations:
0, 0.3, 1.0, 3.0 ml/kg bw/day (0, 252, 840, 2520 mg/kg/day)
Basis:

No. of animals per sex per dose:
25 females per dose group in the main test;
8 females per dose group in the preliminary test
Control animals:
yes, sham-exposed
Details on study design:
- Dose selection rationale: based on the results of the preliminary test
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: before study initiation

BODY WEIGHT: Yes
- Time schedule for examinations: GD 0, 6, 9, 12, 15, and 21

FOOD CONSUMPTION: yes.
- Food consumption measured for each 3-day interval from GD 0 to 21

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): n.a.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 21
- Organs examined: weights of uterus, liver, spleen, adrenals, kidneys, and thymus were recorded. Ovaries, cervices, vaginas, and thoracic and abdominal cavities were examined grossly.


Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other:
Fetal examinations:
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: Yes: cranofacial half per litter
Statistics:
Levene's test for equal variances, ANOVA, and t-tests with Bonferroni probabilities for pairwise comparaison were used. Nonparametrid data were evaluated using the Kruskal-Wallis test followed by Mann-Whitney test when appropriate. Incidences wre compared using Fisher's exact test
Historical control data:
Not required
Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
Administration of 2-ethylhexanol by occluded cutaneous application to time-pregnant Fischer 344 rats during organogenesis at 0, 0.3, 1.0, or 3.0 ml/kg bw/day (25 animals per dose) resulted in maternal toxicity at 1.0 and 3.0 ml/kg/day (clinical signs of toxicity at the dosing site for both doses and reduced weight gain in the treatment period at 3.0 ml/kg/day).
Dose descriptor:
NOAEL
Effect level:
840 mg/kg bw/day
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOAEL
Effect level:
2 520 mg/kg bw/day
Basis for effect level:
other: developmental toxicity
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
There was no developmental toxicity at any test dose. There was no treatment-related increased incidence of malformations.
Dose descriptor:
NOAEL
Effect level:
2 520 mg/kg bw/day
Basis for effect level:
other: teratogenicity
Abnormalities:
not specified
Developmental effects observed:
not specified
Maternal data
Fate:
No females died, aborted or were removed from the study. Two sham control and 
two low dose rats delivered early, and their data were omitted (table 1). Of the treated
rats thge pregnancy rates ranged between 88 to 92%; the viability was 100% at 
all dose levels (table 1):
    
 
Dose (mg/kg bw/d)
0
(sham)
252
840
2520
Females in study
25
25
25
25
Delivered
2
2
0
0
Necropsied
- nonpregnant
3
4
2
5
- pregnant 
20
19
23
20
- with only nonviable implants
2
0
0
0
- with viable fetuses
18
19
23
20
% pregnant
Clinical findings for 2 -EH treated rats were limited to lower body weight changes at the beginning of the treatment (GD 6 -9; p<0.05 in the high dose group), skin irriation (exfoliation, encrustation, and erythema, but no edema). Erythema were seen at 840 mg/kg bw/day and above. Skin irritation was generally mild, max. Draize score was 0.3 on GD14 at 1680 mg/kg bw/day in the main study. Nasal and ocular effects (encrustation and discharge) were seen with 2 -EH and sham controls. This effect was therefore not regarded to be treatment-related. Necropsy findings: Residual exfoliation and crusting at the application site in mid- and high-dose 2 -EH groups were the only treatment-related findings; i.e. body weight, gravid uterus weight, and organ weights were all comparable to the controls. Gestational parameters: 2 -EH was without adverse effect, at any treatment level, compared with controls (table 2):
 
Dose levels (mg/kg bw/day)
0 (sham)
252
840
2520
No. pregnant dams
11.6 (a)
10.4
11.3
10.8
Corpora lutea /dam
16.0
15.4
15.7
15.3
Total implants
5.9
6.7
8.3
7.4
Viable implants
4.24
4.36
3.96
3.2
Nonviable implants
0.4
0.2
0.1
0.1
Early resorptions
0.4
0.2
0.1
0.1
Late resorptions
0
0
0
0
Dead fetuses
0
0
0.1
0
Percentage of live fetuses
86
96.8
97.8
99
Sex ratio (% males)
62.8
41.8
43.7
53.4
Fetal body weight (g)
4.59
4.51
4.4
4.5
(a) n = 18
Fetal data Fetal body weight was not affected at any dose level (table 2). The incidence and the pattern of malformations or variations was not changed in treated rats compared to controls (table 3):
 
 
Dose levels (mg/kg bw/day)
0 (sham)
252
840
2520
External malformations
Number with malformations/Number examined
0/110

0/124

0/185

0/147

%
0

0

0

0

Soft tissue malformations
Number with malformations /Number examined
0/110
0/124
0/185
0/147
%
0
0
0
0
Skeletal malformations 
Number with malformations /Number examined
0/110
0/124
0/185
0/147
%
0
0
0
0
External variations 
Number with variations/Number examined
13/110
19/124
37/185
21/147
%
11.8
15.3
20
14.3
Soft tissue variations 
Number with variations/Number examined
30/63
36/66
57/97
42/79
%
47.6
54.5
58.8
53.2
Skeletal variations 
Number with variations/Number examined
53/53
88/88
68/68
31/31
%
100
100
100
100
 
Conclusions:
No developmental toxicity by dermal route noted at and below dose levels producing maternal toxicity.
Executive summary:

The developmental toxicity of 2 -EH following dermal absorption was examined in a OECD TG 414 rat study that was conducted under GLP. 2 -EH was applied to the skin of 25 females at 252, 840, and 2520 mg/kg bw/day under an occlusive dressing during gestational days 6 -15 for 6 hours per day. The dose levels were selected based on the results of a preliminary study (Tyl et al., 1992).

The maternal toxicity was mild. There were no deaths or severe clinical signs of toxicity. A reduced body weight gain in high-dose rats was noted, and local skin irritation in rats at the intermediate and the high dose level.

2 -EH had no adverse effect on the maternal gestational parameters, or maternal organ weights, or on the fetal weight, sex ratio, viability, or the incidence of malformations and variations.

Therefore, the NOAEL for maternal systemic toxicity was 840 mg/kg bw/day, based on the effects on body weight gain; the NOAEL for skin irritation was 252 mg/kg bw/day. The NOAEL for developmental toxicity and teratogenicity was 2520 mg/kg bw/day.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
191 mg/kg bw/day
Study duration:
subacute
Species:
mouse
Quality of whole database:
Several reliable developmental studies in rats and mice using various routes of application are available. These studies are at least similar to testing guidelines, mostly performed according to GLP and of high reliability (Klimisch scores 1 and 2). The overall quality of the database is therefore high. The hazard conclusion was reached based on a weight of evidence approach.
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEC
850 mg/m³
Study duration:
subacute
Species:
rat
Quality of whole database:
Several reliable developmental studies in rats and mice using various routes of application are available. These studies are at least similar to testing guidelines, mostly performed according to GLP and of high reliability (Klimisch scores 1 and 2). The overall quality of the database is therefore high. The hazard conclusion was reached based on a weight of evidence approach.
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
2 520 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
Several reliable developmental studies in rats and mice using various routes of application are available. These studies are at least similar to testing guidelines, mostly performed according to GLP and of high reliability (Klimisch scores 1 and 2). The overall quality of the database is therefore high. The hazard conclusion was reached based on a weight of evidence approach.
Additional information

Oral:

2-EH was examined in a mouse feeding study for its potential for developmental toxicity equivalent to OECD TG 414 and under GLP conditions. Timed pregnant female CD-1 Swiss mice (28 animals/group, body weight range 32.5 to 31.6 g) received 2-EH in the diet at nominal concentrations of 0, 0.009, 0.03, and 0.09% during gestation days 0-17. The animals were housed singly and observations for clinical signs were made daily. Body weights were recorded on gestational day 0, 3, 6, 9, 12, 15, 17. Food consumption and test compound intake were calculated individually. Test substance purity and concentration in the diets was verified using gas chromatography. Test substance purity was >99%. Concentration in the diets was within 99-108% of the nominal concentration.

Maternal effects:

Food intake and hence dose levels were higher than expected. Average intakes were 0, 17, 59, and 191 mg/kg bw/day, respectively. No dams died, delivered early or were removed from study. Pregnancy rates were high (93-96%) and equivalent across all groups. One litter at 0% was fully resorbed; all other pregnant animals had live litters at scheduled necropsy. The numbers of live litters evaluated were 27 at 0.009 and 0.03% and 26 at 0 and 0.09% 2-EH.

There was no treatment-related maternal toxicity observed in this study. Maternal body weights, weight gains (absolute or corrected for gravid uterine weight), gravid uterine weight and liver weight (absolute or relative to body weight) were unaffected. Food consumption (g/kg/day and g/day) was significantly increased for GD 0-3 at 0.09% and unaffected for all other time points evaluated. The calculated consumption of 2-EH, based on gestational food consumption was 0 (0 mmol/kg), 17 (0.13 mmol/kg), 59 (0.46 mmol/kg) and 191 mg/kg/day (1.49 mmol/kg), for the 0, 0.009, 0.03 and 0.090% groups, respectively.

Fetal effects:

There were no effects of exposure to dietary 2-EH on any of the gestational parameters. The number of corpora lutea, uterine implantation sites (live, dead, resorbed), pre- and postimplantation loss, sex ratio and live fetal body weight per litter (all fetuses or separately by sex) were all equivalent across all groups. There were also no treatment-related changes in the incidence of individual, external, visceral, skeletal or total malformations or variations. 

In conclusion, there were no maternal or developmental toxic effects of 2-EH dietary exposure throughout gestation at any concentration tested, in contrast to the qualitatively similar maternal and developmental toxicity previously reported for DEHP (Tyl et al., 1988; not presented here, cf. respective substance dossier) and MEHP (NTP, 1990; not presented here, cf. respective substance dossier) at approximately equimolar doses administered under comparable experimental conditions. The present study therefore indicates that 2-EH plays essentially no role in the expression of DEHP-induced maternal and developmental toxicity. The NOAEL for maternal toxicity and for developmental toxicity and teratogenicity was therefore 191 mg/kg bw/day, the highest dose level tested. This study was conducted equivalent to OECD TG 414 and under GLP conditions, and it is considered to be valid without restriction (Tyl et al., 1991 corresponds to IUCLID study record NTP, 1991).

 

The developmental toxicity of 2 -EH was furthermore investigated in a comparative and thus limited toxicity study according to OECD TG 414 study. The study addressed all the endpoints required in the OECD TG 414 and was conducted under GLP, but used low rat numbers (due to the fact that the study purpose was a comparative screening for several alcohols; only 10 instead of 20 pregnant females). This invalidates the study to some extent, but it provides weight of evidence for the developmental toxicity endpoint. Animals were treated on GD6 to 15 via gavage at concentrations of 130, 650, and 1300 mg/kg bw/d. Controls were either treated with water only or with vehicle (i.e. 0.005% Cremophor EL).

Maternal toxicity was most severe at the high dose level including mortality (6/10 animals). In the mid and low dose no relevant maternal toxicity was noted. Therefore the NOAEL is 650 mg/kg/d for this endpoint under the conditions of this study.

Signs of embryo-/fetotoxicity were dose-dependently noted in dams showing signs of maternal toxicity at 1300 mg/kg/d. The observed effects (decreased mean fetal bw, increase of skeletal variations and retardation) in the mid-dose group were invalidated when compared to historical controls or current expert opinion in agreement with Regulation (EC) No 1272/2008 was applied:

- the decreased body weights were within the range of historical controls

- original statistical evaluation of the study was assessed to be invalid (reevaluation was performed)

- within this statistical reevaluation (Wilcoxon test; based on litter data) the statistical significance of skeletal variations was not confirmed

- skeletal retardations were still significant

- however skeletal retardations of control goups also exceeded the historical control values

- and in agreement with current expert opinion and Regulation (EC) No 1272/2008 skeletal retardations can be considered as transient non-adverse effects.

Therefore the NOAEL is 650 mg/kg/d for this endpoint under the conditions of this study.

Teratogenicity was noted in fetuses from the high dose dams only. Therefore the NOAEL is 650 mg/kg/d under the conditions of this study for teratogenicity (BASF AG, 1997).

Dermal:

The developmental toxicity of 2-EH following dermal absorption was examined in a OECD TG 414 rat study that was conducted under GLP. 2-EH was applied to the skin of 25 females at 0, 252, 840, and 2520 mg/kg bw/day under an occlusive dressing during gestational days 6 -15 for 6 hours per day. The dose levels were selected based on the results of a preliminary study.

The maternal toxicity was mild. There were no deaths or severe clinical signs of toxicity. A reduced body weight gain in high-dose rats was noted and local skin irritation in rats at the intermediate and the high dose level.

2-EH had no adverse effect on the maternal gestational parameters, or maternal organ weights, or on the fetal weight, sex ratio, viability, or the incidence of malformations and variations.

Therefore, the NOAEL for maternal systemic toxicity was 840 mg/kg bw/day, based on the effects on body weight gain; the NOAEL for skin irritation was 252 mg/kg bw/day. The NOAEL for developmental toxicity and teratogenicity was 2520 mg/kg bw/day (Tyl et al., 1992).

Inhalation:

No maternal toxicity or developmental toxicity was noted in a rat inhalation study that was conducted similar to OECD TG 414; the study is valid though less animals (n = 15) than suggested by the test guideline. The rats were exposed during days 0-19 of gestation to 2-EH at 850 mg/m³.

There were only minor signs of maternal toxicity, i.e. reduction of feed consumption (p<0.05) and body weight gain during gestation. The reproduction parameters were unchanged. There was no indication of developmental toxicity or teratogenicity, as the incidences of resorptions, the number of fetuses per litter, the sex ration, fetal weight, were all comparable to the control group. Moreover, there were no external, skeletal or visceral malformations observed in any group.

The authors concluded that due to the low vapor pressure the systemic dose was too low to produce maternal or developmental toxicity. This does not only apply for 2-EH, but also for other alcohols with chain lengths of 5 or more carbons. Smaller alcohols with chain lengths of 1 to 4 carbons are much more volatile, and produce maternal toxicity, and also developmental toxicity in the presence of maternal toxicity.

The inhalation NOAEC for maternal toxicity and developmental toxicity and teratogenicity was therefore 850 mg/m³ (Nelson et al., 1989).

REMARK:

Other oral or inhalation developmental toxicity studies are judged as invalid due to extreme maternal mortality, major methodological and/or reporting deficiencies (Hardin et al., 1987; Ritter et al., 1987; NTP, 2007a). These studies are considered invalid and do not contribute to the overall hazard assessment concerning developmental toxicity.


Justification for selection of Effect on developmental toxicity: via oral route:
One reliable developmental toxicity study for the oral exposure route (Klimisch score 1)

Justification for selection of Effect on developmental toxicity: via inhalation route:
One reliable developmental toxicity study for the inhalation exposure route (Klimisch score 2)

Justification for selection of Effect on developmental toxicity: via dermal route:
One reliable developmental toxicity study for the dermal exposure route (Klimisch score 1)

Justification for classification or non-classification

Taken from the evidence provided within the two generation study performed with DEHT (precursor of the submission substance 2 - EH) no adverse effects on reproduction are attributed to 2 -EH. Moreover supportive studies indicate that there are no effects of 2 -EH on the gestational parameters of dams in developmental studies, and no effects on the testes can be atttributed to 2 -EH.

Based on the data provided by reliable studies with different administration pathways and in different rodent species, it is concluded that 2-EH exhibited no adverse developmental effect in the absence of maternal toxicity. Overall no classification for reproduction or developmental toxicity has to be performed according to EC Directive 67/548 or Regulation (EC) no 1272/2008.