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 based on grouping of substances (category approach)
Adequacy of study:
key study
Study period:
17 Nov 2003 - 18 Jul 2004
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP-Guideline study with acceptable restrictions. Lack of test material details.
Qualifier:
according to guideline
Guideline:
OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
Deviations:
yes
Remarks:
lack of test material details.
GLP compliance:
yes (incl. QA statement)
Remarks:
Freie und Hansestadt Hamburg, Behörde für Wissenschaft und Gesundheit, Hamburg, Germany
Limit test:
no
Species:
rat
Strain:
other: CD® / Crl:CD®
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland GmbH, Sulzfeld, Germany
- Age at study initiation: (P) 40-47 days (males/females)
- Weight at study initiation: (P) 164.0 - 206.3 g (males) and 139.9 - 176.1 g (females)
- Housing: pre-mating animals and the pregnant females were housed individually, the rearing females were housed together with their pups and the mating animals were kept as pairs in Makrolon type III cages. Coarse granulated wood (Brandenburg, Goldenstedt-Arkeburg, Germany) was used as bedding material.
- Diet: ssniff R-Z V1324 (ssniff Spezialdiäten GmbH, Soest, Germany), ad libitum
- Water: tap water, ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Humidity (%): 55 ± 15
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: once daily, 7 days/week

VEHICLE
- Amount of vehicle: 2 mL/kg bw
- Lot/batch no.: 31107453
Details on mating procedure:
- M/F ratio per cage: 1/1
- Length of cohabitation: until copulation occurred or 2 weeks had elapsed
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as Day 0 of pregnancy
- After 14 days of unsuccessful pairing the animals were separated without further opportunity for mating.
- After successful mating each pregnant female was caged: individually
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The determination of the test substance in corn oil mixtures was performed applying a GC method with FID detection. The test substance was quantified applying a reference standard. The following parameters were determined: linearity, accuracy, precision, sensitivity, specificity and stability. The results of the analysis showed that the test item vehicle mixtures were correctly prepared and the concentration and stability found were in good agreement to those expected. The actual concentrations of the test item-carrier mixtures were within the measured range of 97.4-104.7% of the nominal test item concentrations.
Duration of treatment / exposure:
(F0) Males: 10 weeks before mating, 2 weeks during mating.
(F0) Females: 10 weeks before mating, up to 2 weeks during mating, approximately 3 weeks during pregnancy, 3 weeks during lactation.
(F1) Males: 10 weeks before mating, 2 weeks during mating.
(F1) Females: 10 weeks before mating, up to 2 weeks during mating, approximately 3 weeks during pregnancy, 3 weeks during lactation.
Frequency of treatment:
daily, 7 days/week
Details on study schedule:
- F1 parental animals not mated until at least 10 weeks after selected from the F1 litters.
- Selection of parents from F1 generation when pups were 3 weeks of age.
Remarks:
Doses / Concentrations:
100, 300 and 1000 mg/kg bw/day
Basis:
nominal conc.
No. of animals per sex per dose:
20
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels for this study were selected in agreement with the sponsor based on available toxicity data.
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: Parental animals (F0 and F1) were weighed on the first day of dosing and at least weekly thereafter. Parental females (F0 and F1) were weighed daily during gestation and during lactation on the same days as the weighing of litters and on the day the animals were sacrificed.

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption for each animal was determined weekly during the pre-mating period and daily during the gestation periods. After parturition and during lactation, food consumption measurements were performed on the same day as the weighing of the litters.
Oestrous cyclicity (parental animals):
Oestrous cycle length and normality were evaluated in F0 and F1 females by vaginal smear 3 weeks prior to mating until evidence of mating was found. When obtaining vaginal/cervical cells, care was taken to avoid disturbance of mucosa and subsequently, the induction of pseudopregnancy.
Sperm parameters (parental animals):
Parameters examined in [F0/F1] male parental generations: testis weight, epididymis weight and one of each paired organ was reserved for histopathological examination. Of a subset of at least 10 males of each group, the remaining testes and epididymides were used for enumeration of ultra-sound resistant (homogenisation-resistant) spermatids and cauda epididymides sperm reserves, respectively. From the same groups, sperm motility and sperm morphology was determined.
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: no

PARAMETERS EXAMINED
The following parameters were examined in [F1 / F2 ] offspring: number and sex of pups, weight, presence of milk in the stomach, externally visible abnormalities, stillbirths.
- Functional development: mid-air righting reflex, auditory startle reflex, pupillary reflex, open field, passive avoidance learning and memory.
- Physical development: pinna detachment, ear and eye opening, cleavage of the balanopreputial gland, vaginal opening, upper incisor eruption.

GROSS EXAMINATION OF DEAD PUPS:
yes, all pups with external abnormalities or clinical signs and at least one randomly selected pup/sex/litter from F1 and F2 generation, were examined macroscopically for any structural abnormalities or pathological changes.
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals at the end of the mating period
- Maternal animals: All surviving animals after 3 lactation weeks

GROSS NECROPSY
- Gross necropsy consisted of external surfaces, orifices, cranila cavity, carcass and all organs. Special attention was paid to the organs of the reproductive system.

HISTOPATHOLOGY / ORGAN WEIGHTS
The following tissues were prepared for microscopic examination: adrenal glands, coagulation gland, epididymides, grossly abnormal tissue, ovary, pituitary, prostate, seminal vesicle, testicle, uterus (with oviducts and cervix) and vagina.
The following organs were weighed: adrenal glands, brain, epididymides, kidney, liver, ovary, pituitary, prostate, seminal vesicle, spleen, testicle, thyroid and uterus (with oviducts and cervix).
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring not selected as parental animals or selected for open field and passive avoidance testes and all F2 offspring were sacrificed after 3 lactation weeks.
- These animals were subjected to postmortem examinations macroscopic and microscopic examination as follows: full histopathology of the organs were performed for all high dose and control F1 parental animals per sex.

GROSS NECROPSY
- Gross necropsy consisted of grossly abnormal tissue.

HISTOPATHOLOGY
- Grossly abnormal tissue from all pups with external abnormalities or clinical signs, as well as from at least one randomly selected pup/sex/litter from F1 and F2 generation were fixed for possible histopathological examinations. In F1 females evaluation of Corpora lutea of lactation in ovaries was performed.
Statistics:
For all numerical values homogeneity of variances was tested by using the BARTLETT chi-square test. If the variances were homogenous, the DUNNETT test (p ≤ 0.01) was used to compare the experimental groups with the control group.
In case of heterogeneity of variances, the STUDENT´s t-test was carried out, limit of significance was p ≤ 0.01.
For the comparison of classification measurements the FISHER´s exact test (≤ 0.05), n < 100 or chi²-test with Yates´correction for continuity, n ≥ 100 (p ≤ 0.01) was employed.
Reproductive indices:
For each group the following calculations were performed:
- Fertility female index: (number of pregnancies/number of matings) x 100
- Gestation index: (number of litters with live pups/number of pregnancies) x 100

For each litter and group the following calculations were performed:
- Live-born index: (number of pups born alive/total number of pups born) x 100
- Lactation index: (number of pups alive on Day 21/number of pups alive on Day 4) x 100
- Sex ratio: number of males/number of females
- Percentage by Sex: number of males (females)/total number of animals x 100
- Viability index: (number of pups alive on Day 4/number of pups alive and kept on Day 0) x 100
Offspring viability indices:
For each litter and group the following calculations were performed:
- Live-born index: (number of pups born alive/total number of pups born) x 100
- Lactation index: (number of pups alive on Day 21/number of pups alive on Day 4) x 100
- Sex ratio: number of males/number of females
- Percentage by Sex: number of males (females)/total number of animals x 100
- Viability index: (number of pups alive on Day 4/number of pups alive and kept on Day 0) x 100
Clinical signs:
no effects observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
F0: Low-/mid-dose (f): increased body weight gain; high-dose (f): decreased body weight gain, low-dose (f): increased food consumption, non-adverse; F1: mid-dose (f): decreased body weight; high-dose (f): increased body weight, non-adverse
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
F0: Low-/mid-dose (f): increased body weight gain; high-dose (f): decreased body weight gain, low-dose (f): increased food consumption, non-adverse; F1: mid-dose (f): decreased body weight; high-dose (f): increased body weight, non-adverse
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
no effects observed
Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
effects observed, treatment-related
Description (incidence and severity):
F0: mid- and high dose: reduced live-born indices; high-dose: increased post-implantation loss and prolonged gestation length, non-adverse; F1: high-dose (f): increased fertility index, non-adverse
CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
F0: No mortality and no signs of systemic toxicity were observed.
F1: No mortality and no signs of systemic toxicity were observed.

BODY WEIGHT (PARENTAL ANIMALS)
F0: No influence was noted on the body weight and the body weight gain of the male rats during the pre-mating and mating period. Statistically significant increase in body weight gain during the pre-mating and mating period of the low- mid-dose female group were observed. Additionally, decrease of body weight gain during the gestation period was observed in the mid-dose group. The differences were not considered to be test item-related (Table 3 under "Any other information on results incl. tables").
F1: No influence was noted on the body weight and the body weight gain of the male rats during the pre-mating and mating period. In the mid-dose female group a decreased body weight gain and in the high and low-dose female group an increased body weight gain was apparent but not considered to be test-item related. A statistically significant increase in food consumption in the low- and high-dose female group was determined and was not considered to be test item-related.

FOOD CONSUMPTION (PARENTAL ANIMALS)
F0: No test item-related influence was observed on the food consumption or the food efficiency of the male animals treated with 100, 300 or 1000 mg/kg bw/day during the pre-mating period. A statistically significant increase in food consumption in the low dose female group was determined and was not considered to be test item-related.
F1: No test item-related influence was observed on the food consumption or the food efficiency of the male animals treated with 100, 300 or 1000 mg/kg bw/day during the pre-mating period.
No test item-related influence was observed on the food consumption or the food efficiency of the female animals treated with 100, 300 or 1000 mg/kg bw/day during the pre-mating, gestation or lactation period. Statistically significant increases in food consumption were observed in the high and low dose group in week 8 and week 20, respectively and were not considered to be test item-related.

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
F0: No test item-related influence on the number or the length of the oestrous cycles was observed at any of the tested dose levels during the pre-mating and the mating period. No test item-related difference was noted for the number of abnormal cycles between the dose level groups and the control group.
F1: No test item-related influence on the number or the length of the oestrous cycles was observed at any of the tested dose levels during the pre-mating and the mating period. No test item-related difference was noted tor the number of abnormal cycles between the dose level groups compared to the control group.

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
F0: No test item-related influence was noted on the male fertility of the animals.
The number of ultrasound-resistant spermatids per gram testicular tissue was not influenced at any of the tested dose levels. No test item related changes were noted in the percentage of motile spermatozoa in the epididymal cauda for the males treated with either 100, 300 or 1000 mg/kg bw/day. The mean percentage of morphologically normal spermatids was not different in the dosed males as compared to the control animals.
F1: No test item-related influence was noted on the male fertility of the animals. The number of ultrasound-resistant spermatids per gram testicular tissue was not influenced. No test item related changes were noted in the percentage of motile spermatozoa in the epididymal cauda for the males treated with either 100, 300 or 1000 mg/kg bw/day. The mean percentage of morphologically normal spermatids was not different in the dosed males as compared to the control animals.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
F0: In the high dose group an increased postimplantation loss (statistically significant at p ≤ 0.05) and a statistically (at p ≤ 0.01) significantly prolonged gestation length was observed. The value of post-implantation loss was slightly out of the range of the historical control data and was not considered to be of toxicological relevance. The marginally and not dose-related prolonged gestation length in the high dose group was 21.8 days compared to 21.2 days of the control group.The slight but statistically (at p ≤ 0.05) significantly reduced live-born indices at 300 or 1000 mg/kg bw/day were regarded to be spontaneous; the values were still within the background data of the testing laboratory. Furthermore, the reduced values in each group are based on one litter each only with no viable pups. In the high dose dam no. 171 revealed only one pup which was dead, in the intermediate dose dam no. 126 revealed a litter with 9 dead pups. If both litters are excluded from calculation, the mean live-born index is 98.8% for the intermediate dose group and 98.6% tor the high dose group. These values are within the range of the control group. No test item-related influence was noted for the number of implantation sites, pups at birth (alive and dead), runts or malformed pups, in the number of dams with runts or malformed pups as well as in the value calculated for the gestation index. No female aborted. No malformed pups ware noted. No test item-related influence was noted on the sex ratio, the percentage by sex and the survival indices (viability indices and lactation index) in any of the tested dose groups (see Table 1 and 5 under "Any other information on results incl. tables").
F1: No test substance-related influence on post-implantation loss and gestation length was observed in any animal.

SEXUAL MATURATION
F1: No test item-related influence was noted on the female fertility index of the animals treated with 100, 300 or 1000 mg/kg bw/day. The statistically significantly (at p ≤ 0.01) increased fertility index of the high dosed females is due to the relatively low fertility index of the control females and, therefore, of no biological relevance (see Table 1). No test item-related influence was noted on the pre-coital time of the females.
There were no test item-related differences between the control group and the treated animals in the number of implantation sites, pups at birth (alive and dead), stillbirths, live born pups, in the number of dams with stillborn pups, runts and malformed pups as well as in the values calculated for the gestation index, the gestation length, the live-born Index and the post-implantation loss. No female aborted.
No test item-related influence was noted on the sex ratio, the percentage by sex and the survival indices (viability indices and lactation index) in any of the tested dose groups.

ORGAN WEIGHTS (PARENTAL ANIMALS)
F0: In the high-dose females a statistically significant increase in relative organs weights of brain, spleen and pituitary were observed. These slight alterations in comparison to the control animals were without biological relevance. In the low dose female group, increased pituitary and uterus weight was observed and not considered to be test item-related. The respective absolute organ weights remained unchanged (see Table 2 under "Any other information on results incl. tables").
F1: No test item-related influence was noted tor the relative and absolute organ weights of male and female animals treated with 100, 300 or 1000 mg/kg bw/day. A slight but statistically significantly (at p ≤ 0.01) reduced relative organ weight of the left adrenal is regarded as spontaneous as the effect was observed for the left adrenal only.

GROSS PATHOLOGY (PARENTAL ANIMALS)
F0: Macroscopic inspections at necropsy revealed no test item-related changes in the organs or tissues of the animals treated with either 100, 300 or 1000 mg/kg bw/day (see Table 2 under "Any other information on results incl. tables").
F1: No test item-related changes were observed during necropsy.

HISTOPATHOLOGY (PARENTAL ANIMALS)
F0: The histomorphological examination of rats treated with the high-dose did not reveal any microscopical morphological changes which are considered to be related to the treatment with the test item. Type, incidence and severity of the lesions recorded were not increased in the treated animals as compared to the control animals. All lesions observed were interpreted as spontaneous organ changes and were thus not test item-related.
F1: The histopathological examination of the high-dose group did not reveal any morphological changes which are considered to be related to the test item. In F1 females unaffected Corpora lutea of lactation were present in ovaries of all females. No statistically significant difference was noted on the number of primordial and small growing follicles during examination of the ovaries.
Dose descriptor:
NOEL
Effect level:
300 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: overall effects
Dose descriptor:
NOAEL
Effect level:
> 1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: Increased post-implantation loss and increased gestation length.
Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
F1 (until weaning) high-dose (m, f): increased body weight
Sexual maturation:
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
F1 (until weaning) low- and high-dose (f, m): decreased relative brain weight, non-adverse
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
F1 (until weaning) low- and high-dose: delayed ear opening; high dose (m, f): delayed pinna detachement and eye opening; mid- and high-dose (f): delayed vaginal opening, non-adverse
Histopathological findings:
no effects observed
CLINICAL SIGNS AND MORTALITY (OFFSPRING)
F1/F2: No clinical signs and no mortality were reported.

DEVELOPMENTAL LANDMARKS (OFFSPRING)
F1: No test item-related influence was noted on the physical development. No delayed development was noted for the examined pinna detachment, upper incisor eruption, ear and eye opening.
Statistically significant differences in morphological landmarks which are not considered to be test item-related are listed in Table 4 under "Any other information on results incl. tables".

BODY WEIGHT (OFFSPRING)
F1: No test item-related influence was noted on the mean and total litter weight at any of the tested dose levels. Statistically significant differences in mean body weight where apparent for the following groups but were not considered to be test item-related: increase in mean body weight of the high dose females and males.
F2: No test item-related influence was noted on the mean and total litter weight at any of the tested dose levels.

ORGAN WEIGHTS (OFFSPRING)
F1: No test item-related influence was noted for the relative and absolute organ weights. A statistically significant (at p ≤ 0.01) decreased relative brain weight of the male and female pups of the low and high dose group is regarded to be due to the slightly increased body weight at autopsy observed for these dose groups.
F2: No test item-related influence was noted for the relative and absolute organ weights of the selected male and female F2 pups at any of the tested dose levels.

GROSS PATHOLOGY (OFFSPRING)
F1: No test item-related changes were observed in the organs or tissues of the F1 pups selected for necropsy.
F2: No test item-related changes were observed in the organs or tissues of the F2 pups selected for necropsy.

SEXUAL MATURATION (OFFSPRING)
F1: No delayed development was noted for the cleavage of balanopreputial gland and vaginal opening.

HISTOPATHOLOGY (OFFSPRING)
F1: No test item-related changes in the organs or tissues of the F1 pubs were observed at any of the tested dose levels.
F2: No test item-related changes in the organs or tissues of the F1 pubs were observed at any of the tested dose levels.

FUNCTIONAL TESTS (OFFSPRING)
F1: No test item-related influence on the functional development of the F1 pups was observed. The number of pups showed no auditory startle reflex, midair righting reflex and pupillary reflex as well as the number of pups failed the passive avoidance test revealed no difference between the test item-treated animals and the control group. No test item-related influence in the open field test was apparent. The statistically significant (at p ≤ 0.01) decreased number of pups failed the passive avoidance learning test observed in the intermediate and high dose group is regarded to be a spontaneous finding. Under the present test conditions a decreased number of pups tailed the passive avoidance learning test compared to the control is of no biological relevance.

MATERNAL CARE (OFFSPRING)
F1: No deficiencies in maternal care were noted for the females treated with the test substance. No inadequate lactation or cannibalism of alive and healthy pups was observed during the 3-week lactation period.

ANOGENITAL DISTANCE (OFFSPRING)
F2: No test item-related influence was noted on the anogenital distance at any of the tested dose levels. The statistically significantly (at p ≤ 0.01) decreased anogenital distance noted for male pups of the intermediate dose group is regarded to be a spontaneous finding as no dose response relationship was observed.
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
>= 1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: overall effects
Dose descriptor:
NOAEL
Generation:
F2
Effect level:
>= 1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: overall effects
Reproductive effects observed:
not specified

Table 1. Table for reproductive toxicity study.

Group /Dose level

Fertility Index in %

F0         F1

No. of males evaluated for fertility

F0                F1

No. of fertile males

F0          F1

Group 1 (control)

83

79

24

24

22

23

Group 2 (100 mg/kg bw/day)

92

79

24

24

23

24

Group 3 (300 mg/kg bw/day)

92

96*

24

24

24

23

Group 4 (1000 mg/kg bw/day)

96*

88

24

24

24

23

*: statistically significant at p ≤ 0.01 (Fisher test)

Table 2. Organ weights of F0 females.

Organ

Group

Increase ↑

Decrease ↓

Brain

Group 4 (1000 mg/kg bw/day)

↑*

Pituitary

Group 2 (100 mg/kg bw/day)

Group 4 (1000 mg/kg bw/day)

↑*

↑*

Spleen

Group 4 (1000 mg/kg bw/day)

↑*

Uterus

Group 2 (100 mg/kg bw/day)

↑*

*: statistically significant at p ≤ 0.01 (Fisher test)

Table 3. Body weight gain.

Period / Day of age

Group

Increase ↑

Decrease ↓

Pre-mating and mating

Group 2 (100 mg/kg bw/day); F0 females

Group 3 (300 mg/kg bw/day); F0 females

↑*

↑*

Gestation

Group 3 (300 mg/kg bw/day); F0 females

*

Day 7, 14, 21

Group 4 (1000 mg/kg bw/day); F1 (until weaning) males and females

*

Week 1-2

Group 3 (300 mg/kg bw/day); F1 (parental animals) females

*

Week 9-10

Group 4 (1000 mg/kg bw/day); F1 (parental animals) females

*

*: statistically significant at p ≤ 0.01 (Fisher test)

Table 4. Examination of morphological landmarks F1 (until weaning).

Parameter

Group

Accelerated ↑

Delayed ↓

Reason

Time point

Pinna detachment

Group 4 (1000 mg/kg bw/day); females and males

*

A

Day of life

Ear opening

Group 2 (100 mg/kg bw/day); females and males

Group 4 (1000 mg/kg bw/day); females and males

*

*

A, B

A

Day of life

Eye opening

Group 4 (1000 mg/kg bw/day); females and males

*

A

Day of life

Vaginal opening

Group 3 (300 mg/kg bw/day); females

Group 4 (1000 mg/kg bw/day); females

*

*

A, B

A

Day after conception

Day of life

*: statistically significant at p ≤ 0.01 (Fisher test)

A: the slight alternation in comparison to control animals is without biological relevance

B: lacking dose dependence

C: effect is due to the relative low or high value observed for the control group

D: effect observed before start of treatment

Table 5. Table for reproductive performance.

 

Group 1 (control)

Group 2

(100 mg/kg bw/day)

Group 3

(300 mg/kg bw/day)

Group 4 (1000 mg/kg bw/day)

Fertility Index in %
F0
F1


83
79


92
79


92
96*


96*
88

No. of males evaluated for fertility
F0
F1


24
24


24
24


24
24


24
24

No. of fertile males
F0
F1


22
23


23
24


24
23


24
23

Gestation length (days)
F0
F1


21.2
21.2


21.5
21.5


21.5
21.2


21.8##
21.5

Live-Born Index (%)
F0
F1


99.7
100


96.5
100


93.9*
100


93.7*
99.5

Post-implantation loss (%)
F0
F1


 8.0
7.6


15.9
6.1


14.8
7.6


18.9*
6.7

Gestation Index (%)
F0
F1

100
100

100
100

100
100

100
100

Implantation sites (%)
F0
F1


339
325


334
312


299
324


310
341

*: statistically significant at p ≤ 0.05 (Fisher test)

##: statistically significant at p ≤ 0.01 (Dunnett test or Student´s t-test)

Table 6. Maternal effects.

Parameter

Group 1

Control

Group 2

100 mg/kg bw/day

Group 3

300 mg/kg bw/day

Group 4

1000 mg/kg bw/day

Total treated

F0/F1

 

20

 

20

 

20

 

20

No. (%) dead F0/F1

0 (0)

0 (0)

0 (0)

 

0 (0)

 

Body weight gain [g]

mean (Day 1-21)

F0

F1

 

 

53.3 ± 6.4

56 ± 8.1

 

 

53.1 ± 6.4

54 ± 6.9

 

 

51.1 ± 7.9

51.5 ± 5.4

 

 

50.0 ± 11.7

53.1 ± 6.7

Gravid uterine weight [g/kg bw]

F0

F1

 

1.744 ± 0.277

1.488± 0.544

 

2.281 ± 0.649**

1.354± 0.243

 

1.759 ± 0.464

1.557± 1.592

 

2.167 ± 0.710

1.496± 0.461

No. (%) pregnant

F0

F1

 

83

79

 

92

79

 

92

96*

 

96*

88

* p ≤ 0.05 Fisher test

** p ≤ 0.01 Dunnett test or Student´s t-test

Table 7. Litter response.

Parameter

Group 1

0 mg/kg bw/day

Group 2

100 mg/kg bw/day

Group 3

300 mg/kg bw/day

Group 4

1000 mg/kg bw/day

Implantations

[total number]

F0

F1

 

 

339

325

 

 

334

312

 

 

299

324

 

 

310

341

Implantations F0

[total/number of dams ± SD]

 

17 ± 2.3

 

16.7 ± 2.0

 

15 ± 3.0

 

15.5 ± 3.2

Total number of live and dead foetuses at birth from

F0

F1

 

 

313

300

 

 

286

293

 

 

269

299

 

 

265

320

Total number of dead foetuses

F0

F1

 

1

0

 

8

0

 

12

0

 

5

1

Total number of live born pups

F0

F1

 

312

300

 

278

293

 

257

299

 

260

319

Post-implantation loss(%)

F0

F1

 

8.0

7.6

 

15.9

6.1

 

14.8

7.6

 

18.9*

6.7

Foetal sex rationat birth (mean %, m:f)

F1

F2

 

 

44.7/55.3

50/50

 

 

52.8/47.2

52/48

 

 

50.9/49.1

49/51

 

 

51.4/48.6

51/49

Foetus weight (mean at Day of Age 1) [g]

F1

F2

 

 

6.4

6.5

 

 

6.7

6.7

 

 

6.7

6.6

 

 

6.8

6.8

Number of malformed pups

F1

F2

0

0

0

0

0

0

0

1#

Number of runts

F1

F2

 

1

1

 

2

0

 

0

1

 

0

0

*: statistically significant at p≤ 0.05 (Fisher test)

#: short tail was observed for pup no. 487-5 m

Conclusions:
The test substance had no effect on reproductive performance.
Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Quality of whole database:
The available information comprises an adequate and reliable study (Klimisch score 2) from a reference substance with similar structure and intrinsic properties. Read-across is justified based on common functional group(s), common precursors and breakdown products and similarities in PC/ECO/TOX properties (refer to endpoint discussion for further details). The selected study is thus sufficient to fulfil the standard information requirements set out in Annex VIII-IX, 8.7, in accordance with Annex XI, 1.5, of Regulation (EC) No 1907/2006.
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

Justification for grouping of substances and read-across

The Glycol ester category covers esters of an aliphatic diol (ethylene glycol (EG), propylene glycol (PG) or 1,3-butyleneglycol (1,3-BG)) and one or two carboxylic fatty acid chains. The fatty acid chains comprise carbon chain lengths ranging from C6 to C18, mainly saturated but also mono unsaturated C16 and C18, branched C18 and epoxidized C18. Fatty acid esters are generally produced by chemical reaction of an alcohol (e.g. ethylene glycol) with an organic acid (e.g. stearic acid) in the presence of an acid catalyst (Radzi et al., 2005). The esterification reaction is started by a transfer of a proton from the acid catalyst to the acid to form an alkyloxonium ion. The acid is protonated on its carbonyl oxygen followed by a nucleophilic addition of a molecule of the alcohol to a carbonyl carbon of acid. An intermediate product is formed. This intermediate product loses a water molecule and a proton to give an ester (Liu et al, 2006; Lilja et al., 2005; Gubicza et al., 2000; Zhao, 2000). Di- and/or monoesters are the final products of esterification of an aliphatic diol and fatty acids.

In accordance with Article 13 (1) of Regulation (EC) No 1907/2006, "information on intrinsic properties of substances may be generated by means other than tests, provided that the conditions set out in Annex XI are met. In particular for human toxicity, information shall be generated whenever possible by means other than vertebrate animal tests", which includes the use of information from structurally related substances (grouping or read-across).

Having regard to the general rules for grouping of substances and read-across approach laid down in Annex XI, Item 1.5, of Regulation (EC) No 1907/2006, whereby substances may be considered as a category provided that their physicochemical, toxicological and ecotoxicological properties are likely to be similar or follow a regular pattern as a result of structural similarity, the substances listed below are allocated to the category of Glycol esters.

 

CAS

EC name

Molecular weight

Carbon number in Acid

Carbon number in dihydroxy alcohol

Total Carbons in Glycol Esters

CAS 111-60-4 (b)

Glycol stearate

MW 328.53

C18

C2

C20

CAS 624-03-3 (a)          

Ethane-1,2-diyl palmitate

MW 538.89

C16

C2

C34

CAS 627-83-8               

Ethylene distearate

MW 563.0

C18

C2

C38

CAS 91031-31-1

Fatty acids, C16-18, esters with ethylene glycol

MW 300.48 - 563.00

C16-18

C2

C18-38

CAS 151661-88-0

Fatty acids, C18 and C18 unsatd. epoxidized, ester with ethylene glycol

MW 328.54 - 622.97

C18

C2

C20-38

CAS 29059-24-3

Myristic acid, monoester with propane-1,2-diol

MW 286.45

C14

C3

C17

CAS 1323-39-3

Stearic acid, monoester with propane-1,2-diol

MW 342.55

C18

C3

C21

CAS 37321-62-3

Dodecanoic acid, ester with 1,2-propanediol

MW 258.40 - 440.71

C12

C3

C15-27

CAS 68958-54-3

1-methyl-1,2-ethanediyl diisooctadecanoate

MW 609.03

C18

C3

C39

CAS 31565-12-5

Octanoic acid ester with 1,2-propanediol, mono- and di-

MW 202.29 - 328.49

C8

C3

C11-19

CAS 85883-73-4

Fatty acids, C6-12, esters with propylene glycol

MW 202.29 - 440.71

C6-12

C3

C9-27

CAS 68583-51-7

Decanoic acid, mixed diesters with octanoic acid and propylene glycol

MW 328.49 - 384.59

C8-10

C3

C19-23

CAS 84988-75-0

Fatty acids, C14-18 and C16-18-unsatd., esters with propylene glycol

MW 286.46 - 609.02

C14-18

C3

C17-39

CAS 853947-59-8

Butylene glycol dicaprylate / dicaprate

MW 342.52 - 398.63

C8-10

C4

C20-24

(a) Category members subject to registration are indicated in bold font.

(b) Substances not subject to registration are indicated in normal font.

 

Grouping of substances into this category is based on:

(1) common functional groups: the substances of the category are characterized by ester bond(s) between an aliphatic diol (ethylene glycol (EG), propylene glycol (PG) or 1,3-butyleneglycol (1,3-BG)) and one or two carboxylic fatty acid chains. The fatty acid chains comprise carbon chain lengths ranging from C6 to C18, mainly saturated but also mono unsaturated C16 and C18, branched C18 and epoxidized C18, are included into the category; and

(2) common precursors and the likelihood of common breakdown products via biological processes, which result in structurally similar chemicals: glycol esters are expected to be initially metabolized via enzymatic hydrolysis in the corresponding free fatty acids and the free glycol alcohols such as ethylene glycol and propylene glycol. The hydrolysis represents the first chemical step in the absorption, distribution, metabolism and excretion (ADME) pathways expected to be similarly followed by all glycol esters. The hydrolysis is catalyzed by classes of enzymes known as carboxylesterases or esterases (Heymann, 1980). Ethylene and propylene glycol are rapidly absorbed from the gastrointestinal tract and subsequently undergo rapid biotransformation in liver and kidney (ATSDR, 1997; ICPS, 2001; WHO, 2002; ATSDR, 2010). Propylene glycol will be further metabolized in liver by alcohol dehydrogenase to lactic acid and pyruvic acid which are endogenous substances naturally occurring in mammals (Miller & Bazzano, 1965, Ritchie, 1927). Ethylene glycol is first metabolised by alcohol dehydrogenase to glycoaldehyde, which is then further oxidized successively to glycolic acid, glyoxylic acid, oxalic acids by mitochondrial aldehyde dehydrogenase and cytosolic aldehyde oxidase (ATSDR, 2010; WHO, 2002). The anabolism of fatty acids occurs in the cytosol, where fatty acids esterified into cellular lipids that are the most important storage form of fatty acids (Stryer, 1994). The catabolism of fatty acids occurs in the cellular organelles, mitochondria and peroxisomes via a completely different set of enzymes. The process is termed ß-oxidation and involves the sequential cleavage of two-carbon units, released as acetyl-CoA through a cyclic series of reaction catalyzed by several distinct enzyme activities rather than a multienzyme complex (Tocher, 2003); and

(3) constant pattern in the changing of the potency of the properties across the category:

(a) Physico-chemical properties: The physico-chemical properties of the category members are similar or follow a regular pattern over the category. The pattern observed depends on the fatty acid chain length and the degree of esterification (mono- or diesters). The molecular weight of the category members ranges from 202.29 to 622.97 g/mol. The physical appearance is related to the chain length of the fatty acid moiety, the degree of saturation and the number of ester bonds. Thus, mono- and diesters of short-chain fatty acids and unsaturated fatty acids (C6-14 and C16:1, C18:1) as well as diesters of branched fatty acids (C18iso) are liquid, while mono- and diesters of long-chain fatty acids are waxy solids. All category members are non-volatile (vapour pressure: ≤ 0.066 Pa). The octanol/water partition coefficient increases with increasing fatty acid chain length and number of ester bonds, ranging from log Kow = 1.78 (C6 PG monoester component) to log Kow >10 (C12 PG diester component). The water solubility decreases accordingly (624.3 mg/L for C6 PG monoester component to >0.01 mg/L for C18 PG diester component); and

(b) Environmental fate and ecotoxicological properties: Considering the low water solubility and the potential for adsorption to organic soil and sediment particles, the main compartment for environmental distribution is expected to be the soil and sediment. Nevertheless, persistency in these compartments is not expected since the members of the Glycol Esters Category are readily biodegradable. Evaporation into air and the transport through the atmospheric compartment is not expected since the category members are not volatile based on the low vapour pressure. All members of the category are readily biodegradable and did not show any effects on aquatic organisms in acute and chronic tests representing the category members up to the limit of water solubility. Moreover, bioaccumulation is assumed to be low based on metabolism data.

(c) Toxicological properties: The toxicological properties show that all category members have a similar toxicokinetic behaviour (hydrolysis of the ester bond before absorption followed by absorption and metabolism of the breakdown products) and that the constant pattern consists in a lack of potency change of properties across the category, explained by the common metabolic fate of glycol esters independently of the fatty acid chain length and degree of glycol substitution. Thus, no category member showed acute oral, dermal or inhalative toxicity, no skin or eye irritation properties, no skin sensitisation, are of low toxicity after repeated oral exposure and are not mutagenic or clastogenic and have shown no indications for reproduction toxicity and have no effect on intrauterine development.

The available data allows for an accurate hazard and risk assessment of the category and the category concept is applied for the assessment of environmental fate and environmental and human health hazards. Thus, where applicable, environmental and human health effects are predicted from adequate and reliable data for source substance(s) within the group by interpolation to the target substances in the group (read-across approach) applying the group concept in accordance with Annex XI, Item 1.5, of Regulation (EC) No 1907/2006. In particular, for each specific endpoint the source substance(s) structurally closest to the target substance is/are chosen for read-across, with due regard to the requirements of adequacy and reliability of the available data. Structural similarities and similarities in properties and/or activities of the source and target substance are the basis of read-across.

A detailed justification for the grouping of chemicals and read-across is provided in the technical dossier (see IUCLID Section 13).

Data Matrix

CAS #

Toxicity to reproduction

624-03-3 (a)

RA: CAS 853947-59-8

627-83-8

RA: CAS 853947-59-8

68583-51-7

RA: CAS 853947-59-8

84988-75-0

RA: CAS 853947-59-8

853947-59-8 (b)

NOAEL: 1000 mg/kg bw/day

(a) Category members subject to registration are indicated in bold font. Only for these substances a full set of experimental results and/or read-across is given.

(b) Substances not subject to registration are indicated in normal font. Lack of data for a given endpoint is indicated by “--“.

 

CAS 627-83-3, CAS 68583-51-7, CAS 84988-75-0 and CAS 624-03-3

Within the category of Glycol Esters, one study with Butylene glycol dicaprylate / dicaprate (CAS 853947-59-8) is available. The study of the category member was considered for assessment and read-across was conducted based on a category approach.

Butylene glycol dicaprylate / dicaprate was tested for toxicity to reproduction in a two-generation reproduction toxicity study according to OECD 416 in compliance with GLP (Cordts, 2005, 2005). Groups of 20 CD® / Crl:CD® rats per sex and dose were given 100, 300 and 1000 mg/kg bw/day of the test material by gavage. Males were given the test material daily for 10 weeks until mating and 2 weeks during mating. Females were treated with the test material in the same way and additionally approximately 3 weeks during pregnancy and 3 weeks during lactation. A concurrent negative control group receiving the vehicle corn oil only was included in the testing as well. Examination of the parental animals revealed no clinical signs of toxicity or mortality in relation to the test substance. Furthermore, no influence was noted on the body weight and the body weight gain of the male rats during the pre-mating and mating period. Statistically significant differences in body weight gain in females were observed but were not considered to be test item-related. No influence on food consumption or food efficiency was noted in parental males during the premating period and in females during the premating, gestation and lactation period at any of the tested dose levels. In male animals of the parental F0 and F1 generation, examination showed no test-item related influence on the male fertility period, sperm number, viability and morphology within the treated groups. In female animals, no test item-related influence on female fertility indices was noted. In addition, no influence on the number or the length of the oestrous cycles was observed at any of the tested dose levels during the pre-mating and the mating period and no test item-related difference was noted for the number of abnormal cycles between the treated groups and the control group. However, in females of the high-dose group (F0) an increased but not dose-related post-implantation loss was observed. The value of post-implantation loss was slightly out of the range of the historical control data and was not considered to be of toxicological relevance. Furthermore, in the high-dose group a marginally and not dose-related increased prolonged gestation length was observed (21.8 days compared to 21.2 days of the control group). Post-implantation loss and gestation length in F1 females were not influenced as in the F0 females. No further effects on reproductive performance of F0 and F1 animals were observed and evaluation of the pre-coital time showed no test-item related influence in parental animals, as well. Moreover, no deficiencies in maternal care of the parental animals were noted. The macroscopic examination at necropsy revealed no substance-related differences in the organs and tissues of the parental animals. No changes in absolute and relative organ weights of the parental animals were observed, as well. Histopathological examination revealed no morphological changes which were considered to be test item-related.

Examinations of the F1 and F2 offspring showed no effects on the mean and total litter weight. Furthermore, no effects on the physical development of the F1 offspring were noted in any treated group. The anogenital distance in the F2 pups was not influenced by the test substance. At necropsy, no substance-related differences were noted between control and treated animals in the organs or tissues of the selected animals. Furthermore, the absolute and relative organ weights of male and female F1 and F2 pups was not influenced. Functional tests showed no test item-related differences in the functional development of the F1 offspring.

In summary, under the conditions of the study, the substance had no effect on reproductive performance and the NOAEL for reproduction toxicity of the parental and the F1 and F2 generations is considered to be above 1000 mg/kg bw/day (m, f).

References

Agency for Toxic Substances and Disease Registry (ATSDR) (1997): Toxicological Profile for Propylene Glycol. US Department of Health and Human Services. Atlanta, US.

Agency for Toxic Substances and Disease Registry (ATSDR) (2010): Toxicological Profile for Ethylene Glycol. US Department of Health and Human Services. Atlanta, US.

Gubicza, L., Kabiri-Badr, A., Keoves, E., Belafi-Bako, K. (2000): Large-scale enzymatic production of natural flavour esters in organic solvent with continuous water removal. Journal of Biotechnology 84(2): 193-196.

Heymann, E. (1980): Carboxylesterases and amidases. In: Jakoby, W.B., Bend, J.R. & Caldwell, J., eds., Enzymatic Basis of Detoxication, 2nd Ed., New York: Academic Press, pp. 291-323.Gubicza, L. et al. (2000). Large-scale enzymatic production of natural flavour esters in organic solvent with continuous water removal. Journal of Biotechnology 84(2): 193-196.

International Programme on Chemical Safety (IPCS) (2001): Ethylene Glycol. Poisons Information Monograph. PIM 227.

Lilja, J. et al. (2005). Esterification of propanoic acid with ethanol, 1-propanol and butanol over a heterogeneous fiber catalyst. Chemical Engineering Journal, 115(1-2): 1-12.

Liu, Y. et al. (2006). A comparison of the esterification of acetic acid with methanol using heterogeneous versus homogeneous acid catalysis. Journal of Catalysis 242: 278-286.

Miller, O.N., Bazzano, G. (1965): Propanediol metabolism and its relation to lactic acid -metabolism. Annals of the New York Academy of Sciences 119, 957-973.

Radzi, S.M. et al. (2005). High performance enzymatic synthesis of oleyl oleate using immobilised lipase from Candida antartica. Electronic Journal of Biotechnology 8: 292-298.

Ritchie, A.D. (1927): Lactic acid in fish and crustacean muscle. Journal of Experimental Biology 4, 327-332.

Stryer, L. (1994): Biochemie. 2nd revised reprint, Heidelberg; Berlin; Oxford: Spektrum Akad. Verlag.

Tocher, D.R. (2003): Metabolism and Functions of Lipids and Fatty Acids in Teleost Fish. Reviews in Fisheries Science 11(2), 107-184.

WHO (2002): Ethylene Glycol: Human Health Aspects. Concise International Chemical Assessment Document 45.

Zhao, Z. (2000). Synthesis of butyl propionate using novel aluminophosphate molecular sieve as catalyst. Journal of Molecular Catalysis 154(1-2): 131-135.


Short description of key information:
NOAEL oral (fertility): 1000 mg/kg bw/day (OECD 416, GLP)

Justification for selection of Effect on fertility via oral route:
Hazard assessment is conducted by means of read-across from a structural analogue. The selected study is the most adequate and reliable study based on the identified similarities in structure and intrinsic properties between source and target substance and overall assessment of quality, duration and dose descriptor level (refer to the endpoint discussion for further details).

Effects on developmental toxicity

Description of key information
NOAEL oral (developmental): ≥ 900 mg/kg bw/day (OECD 414, GLP)
Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Quality of whole database:
The available information comprises adequate, reliable (Klimisch score 2) studies from reference substances with similar structure and intrinsic properties. Read-across is justified based on common origin, common precursors and breakdown products of hydrolysis and consistent trends in environmental fate, ecotoxicological and toxicological profile (refer to endpoint discussion for further details). The selected studies are thus sufficient to fulfil the standard information requirements set out in Annex VIII-IX, 8.6, in accordance with AnnexXI, 1.5, of Regulation (EC) No 1907/2006.
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

Justification for grouping of substances and read-across

The Glycol ester category covers esters of an aliphatic diol (ethylene glycol (EG), propylene glycol (PG) or 1,3-butyleneglycol (1,3-BG)) and one or two carboxylic fatty acid chains. The fatty acid chains comprise carbon chain lengths ranging from C6 to C18, mainly saturated but also mono unsaturated C16 and C18, branched C18 and epoxidized C18. Fatty acid esters are generally produced by chemical reaction of an alcohol (e.g. ethylene glycol) with an organic acid (e.g. stearic acid) in the presence of an acid catalyst (Radzi et al., 2005). The esterification reaction is started by a transfer of a proton from the acid catalyst to the acid to form an alkyloxonium ion. The acid is protonated on its carbonyl oxygen followed by a nucleophilic addition of a molecule of the alcohol to a carbonyl carbon of acid. An intermediate product is formed. This intermediate product loses a water molecule and a proton to give an ester (Liu et al, 2006; Lilja et al., 2005; Gubicza et al., 2000; Zhao, 2000). Di- and/or monoesters are the final products of esterification of an aliphatic diol and fatty acids.

In accordance with Article 13 (1) of Regulation (EC) No 1907/2006, "information on intrinsic properties of substances may be generated by means other than tests, provided that the conditions set out in Annex XI are met. In particular for human toxicity, information shall be generated whenever possible by means other than vertebrate animal tests", which includes the use of information from structurally related substances (grouping or read-across).

Having regard to the general rules for grouping of substances and read-across approach laid down in Annex XI, Item 1.5, of Regulation (EC) No 1907/2006, whereby substances may be considered as a category provided that their physicochemical, toxicological and ecotoxicological properties are likely to be similar or follow a regular pattern as a result of structural similarity, the substances listed below are allocated to the category of Glycol esters.

 

CAS

EC name

Molecular weight

Carbon number in Acid

Carbon number in dihydroxy alcohol

Total Carbons in Glycol Esters

CAS 111-60-4 (b)

Glycol stearate

MW 328.53

C18

C2

C20

CAS 624-03-3 (a)          

Ethane-1,2-diyl palmitate

MW 538.89

C16

C2

C34

CAS 627-83-8               

Ethylene distearate

MW 563.0

C18

C2

C38

CAS 91031-31-1

Fatty acids, C16-18, esters with ethylene glycol

MW 300.48 - 563.00

C16-18

C2

C18-38

CAS 151661-88-0

Fatty acids, C18 and C18 unsatd. epoxidized, ester with ethylene glycol

MW 328.54 - 622.97

C18

C2

C20-38

CAS 29059-24-3

Myristic acid, monoester with propane-1,2-diol

MW 286.45

C14

C3

C17

CAS 1323-39-3

Stearic acid, monoester with propane-1,2-diol

MW 342.55

C18

C3

C21

CAS 37321-62-3

Dodecanoic acid, ester with 1,2-propanediol

MW 258.40 - 440.71

C12

C3

C15-27

CAS 68958-54-3

1-methyl-1,2-ethanediyl diisooctadecanoate

MW 609.03

C18

C3

C39

CAS 31565-12-5

Octanoic acid ester with 1,2-propanediol, mono- and di-

MW 202.29 - 328.49

C8

C3

C11-19

CAS 85883-73-4

Fatty acids, C6-12, esters with propylene glycol

MW 202.29 - 440.71

C6-12

C3

C9-27

CAS 68583-51-7

Decanoic acid, mixed diesters with octanoic acid and propylene glycol

MW 328.49 - 384.59

C8-10

C3

C19-23

CAS 84988-75-0

Fatty acids, C14-18 and C16-18-unsatd., esters with propylene glycol

MW 286.46 - 609.02

C14-18

C3

C17-39

CAS 853947-59-8

Butylene glycol dicaprylate / dicaprate

MW 342.52 - 398.63

C8-10

C4

C20-24

 

(a) Category members subject to registration are indicated in bold font.

(b) Substances not subject to registration are indicated in normal font.

 

Grouping of substances into this category is based on:

(1) common functional groups: the substances of the category are characterized by ester bond(s) between an aliphatic diol (ethylene glycol (EG), propylene glycol (PG) or 1,3-butyleneglycol (1,3-BG)) and one or two carboxylic fatty acid chains. The fatty acid chains comprise carbon chain lengths ranging from C6 to C18, mainly saturated but also mono unsaturated C16 and C18, branched C18 and epoxidized C18, are included into the category; and

(2) common precursors and the likelihood of common breakdown products via biological processes, which result in structurally similar chemicals: glycol esters are expected to be initially metabolized via enzymatic hydrolysis in the corresponding free fatty acids and the free glycol alcohols such as ethylene glycol and propylene glycol. The hydrolysis represents the first chemical step in the absorption, distribution, metabolism and excretion (ADME) pathways expected to be similarly followed by all glycol esters. The hydrolysis is catalyzed by classes of enzymes known as carboxylesterases or esterases (Heymann, 1980). Ethylene and propylene glycol are rapidly absorbed from the gastrointestinal tract and subsequently undergo rapid biotransformation in liver and kidney (ATSDR, 1997; ICPS, 2001; WHO, 2002; ATSDR, 2010). Propylene glycol will be further metabolized in liver by alcohol dehydrogenase to lactic acid and pyruvic acid which are endogenous substances naturally occurring in mammals (Miller & Bazzano, 1965, Ritchie, 1927). Ethylene glycol is first metabolised by alcohol dehydrogenase to glycoaldehyde, which is then further oxidized successively to glycolic acid, glyoxylic acid, oxalic acids by mitochondrial aldehyde dehydrogenase and cytosolic aldehyde oxidase (ATSDR, 2010; WHO, 2002). The anabolism of fatty acids occurs in the cytosol, where fatty acids esterified into cellular lipids that are the most important storage form of fatty acids (Stryer, 1994). The catabolism of fatty acids occurs in the cellular organelles, mitochondria and peroxisomes via a completely different set of enzymes. The process is termed ß-oxidation and involves the sequential cleavage of two-carbon units, released as acetyl-CoA through a cyclic series of reaction catalyzed by several distinct enzyme activities rather than a multienzyme complex (Tocher, 2003); and

(3) constant pattern in the changing of the potency of the properties across the category:

(a) Physico-chemical properties: The physico-chemical properties of the category members are similar or follow a regular pattern over the category. The pattern observed depends on the fatty acid chain length and the degree of esterification (mono- or diesters). The molecular weight of the category members ranges from 202.29 to 622.97 g/mol. The physical appearance is related to the chain length of the fatty acid moiety, the degree of saturation and the number of ester bonds. Thus, mono- and diesters of short-chain fatty acids and unsaturated fatty acids (C6-14 and C16:1, C18:1) as well as diesters of branched fatty acids (C18iso) are liquid, while mono- and diesters of long-chain fatty acids are waxy solids. All category members are non-volatile (vapour pressure: ≤ 0.066 Pa). The octanol/water partition coefficient increases with increasing fatty acid chain length and number of ester bonds, ranging from log Kow = 1.78 (C6 PG monoester component) to log Kow >10 (C12 PG diester component). The water solubility decreases accordingly (624.3 mg/L for C6 PG monoester component to >0.01 mg/L for C18 PG diester component); and

(b) Environmental fate and ecotoxicological properties: Considering the low water solubility and the potential for adsorption to organic soil and sediment particles, the main compartment for environmental distribution is expected to be the soil and sediment. Nevertheless, persistency in these compartments is not expected since the members of the Glycol Esters Category are readily biodegradable. Evaporation into air and the transport through the atmospheric compartment is not expected since the category members are not volatile based on the low vapour pressure. All members of the category are readily biodegradable and did not show any effects on aquatic organisms in acute and chronic tests representing the category members up to the limit of water solubility. Moreover, bioaccumulation is assumed to be low based on metabolism data.

(c) Toxicological properties: The toxicological properties show that all category members have a similar toxicokinetic behaviour (hydrolysis of the ester bond before absorption followed by absorption and metabolism of the breakdown products) and that the constant pattern consists in a lack of potency change of properties across the category, explained by the common metabolic fate of glycol esters independently of the fatty acid chain length and degree of glycol substitution. Thus, no category member showed acute oral, dermal or inhalative toxicity, no skin or eye irritation properties, no skin sensitisation, are of low toxicity after repeated oral exposure and are not mutagenic or clastogenic and have shown no indications for reproduction toxicity and have no effect on intrauterine development.

The available data allows for an accurate hazard and risk assessment of the category and the category concept is applied for the assessment of environmental fate and environmental and human health hazards. Thus, where applicable, environmental and human health effects are predicted from adequate and reliable data for source substance(s) within the group by interpolation to the target substances in the group (read-across approach) applying the group concept in accordance with Annex XI, Item 1.5, of Regulation (EC) No 1907/2006. In particular, for each specific endpoint the source substance(s) structurally closest to the target substance is/are chosen for read-across, with due regard to the requirements of adequacy and reliability of the available data. Structural similarities and similarities in properties and/or activities of the source and target substance are the basis of read-across.

A detailed justification for the grouping of chemicals and read-across is provided in the technical dossier (see IUCLID Section 13).

Data Matrix

CAS #

Developmental toxicity

624-03-3 (a)

RA: CAS 853947-59-8

RA: CAS 85883-73-4

RA: CAS 68583-51-7

RA: CAS 91031-31-1

627-83-8

RA: CAS 853947-59-8

RA: CAS 85883-73-4

RA: CAS 68583-51-7

RA: CAS 91031-31-1

91031-31-1 (b)

NOAEL: > 900 mg/kg bw/day

85883-73-4

NOAEL: 1000 mg/kg bw/day

68583-51-7

NOAEL: 1000 mg/kg bw/day

84988-75-0

RA: CAS 853947-59-8

RA: CAS 85883-73-4

RA: CAS 68583-51-7

RA: CAS 91031-31-1

853947-59-8

NOAEL: 1000 mg/kg bw/day

(a) Category members subject to registration are indicated in bold font. Only for these substances a full set of experimental results and/or read-across is given.

(b) Substances not subject to registration are indicated in normal font. Lack of data for a given endpoint is indicated by “--“.

 

CAS 68583-51-7

One study with Decanoic acid, mixed diesters with octanoic acid and propylene glycol is available. The oral prenatal developmental toxicity study was conducted according to OECD 414 under GLP conditions (BASF, 1994).

Groups of 24 female Sprague Dawley rats per dose level were orally dosed with 100, 300 and 1000 mg/kg bw/day by gavage from Day 6-15 of gestation. A concurrent negative control group receiving the vehicle (arachis oil) only was included in the testing as well. No maternal mortality occurred and no substance-related clinical signs of toxicity were observed. In addition, maternal body weight profiles were similar in all groups. Furthermore, at scheduled necropsy no macroscopic changes were noted in maternal animals.

No compound-related differences were observed in pre-and post-implantation loss, embryonic deaths, mean numbers of resorptions and total fetuses of the test groups in comparison to the control group. Mean fetal placental and uterus weights and body weights of live fetus exhibited no significant differences between treatment and control groups. In addition, the fetal sex ratio was not affected by treatment. No treatment-related fetal abnormalities or malformations were found at necropsy. The figures of skeletal and visceral variations in the treatment and control groups were considered to be comparable. Skeletal ossification showed in the low- and high-dose treatment groups one fetus each with incomplete ossified skull bones and additionally one fetus in the high-dose group with non-ossified skull bones. In the control group, 12 fetuses showed incomplete ossified skull bones and 6 fetuses showed non-ossified skull bones as well. Thus, the number of incomplete- and non-ossified skull bones was decreased in the treatment groups in comparison to the control group and the findings were considered to be identical and therefore not treatment-related.

Therefore, due to the lack of adverse effects in this study, the NOAEL for maternal toxicity and developmental toxicity for rats was considered to be 1000 mg/kg bw/day.

 

CAS 624-03-3, CAS 627-83-8 and CAS 84988-75-0

Altogether, four studies investigating the developmental toxicity are available within the Glycol Ester category. The studies from the category members Fatty acids, C16-18, esters with ethylene glycol (CAS 91031-31-1), Decanoic acid, mixed diesters with octanoic acid and propylene (CAS 68583-51-7), C8-10, 1,3-Butandiolester (CAS 853947-59-8) and Fatty acids, C6-12, ester with propylene glycol (CAS 85883-73-4) were considered for assessment and read-across was conducted based on a category and weight of evidence approach.

The results of the study with Decanoic acid, mixed diesters with octanoic acid and propylene are already described above.

Fatty acids, C16-18, esters with ethylene glycol, C8-10, 1,3-Butandiolester and Fatty acids, C6-12, ester with propylene glycol were tested in oral prenatal developmental toxicity studies according to OECD 414 in compliance with GLP (Pittermann, 1997; Cicalese, 2007; Bowman, 2007).

Groups of 24 or 25 female rats per dose were dosed with the respective test compound via gavage from Day 6-15, Day 6-17 or Day 6-19 post mating. Concurrent negative control groups receiving the vehicle alone were included in all testings.

Animals were dosed via gavage with 100, 300 and 900 mg/kg bw/day of Fatty acids, C16-18, esters with ethylene glycol. No mortalities in maternal animals and no compound-related symptoms were observed in the treatment groups. In addition, body weight and body weight gains were within the expected ranges. No compound-related differences were noted between the mean reproduction data of the test groups in comparison to the control group. At scheduled necropsy no macroscopic changes were noted in the dams of the treatment groups. Furthermore, pre-implantation loss, post-implantation loss, mean number of resorptions, embryonic deaths and total fetuses were not affected by treatment with the test substance. In addition, mean fetal placental and uterus weights were not affected. The fetal sex ratio was comparable in all groups and no treatment-related fetal abnormalities were found at necropsy. The examined fetuses showed no treatment-related malformations and the figures of visceral variations in the test groups were considered to be similar to the control group. The mean weight of live male and female fetuses in the mid-dose group was significantly increased, whereas the weights of live fetuses of the other treatment groups exhibited no significant differences. The figures of skeletal ossifications and variations showed no treatment-related deviations; thus various findings, all without dose-relationship, were considered to be incidental.

Based on the lack of adverse effects in this study, the NOAEL for maternal toxicity and developmental toxicity for rats was considered to be above 900 mg/kg bw/day.

 

In the study with C8-10, 1,3-Butandiolester, animals were dosed with the limit dose of 1000 mg/kg bw/day. No mortality or treatment-related signs of toxicity in maternal animals occurred during the study period. Body weight, body weight gain and food consumption were unaffected by treatment. No treatment-related effects in uterus weight or macroscopic changes were detected in treated females. No compound-related differences were observed in pre-and post-implantation loss, embryonic deaths and mean numbers of resorptions of the test groups in comparison to the control group. In fetuses of the treatment group, a slight but statistically significant lower mean fetal weight was observed when compared to the control group but within the historical control data for this rat strain. This slight difference was attributed to the higher presence of fetuses in the treated group compared to controls in which mean fetal weight was also unusually higher when compared to the internal historical control data of the laboratory. Three small fetuses were observed in the control and treatment group and one control fetus and one fetus of the treated group showed multiple anomalies. These changes were considered incidental. Furthermore, spontaneous changes at skeletal examination of the fetuses were noted between the treated and the control group. Visceral examination of fetuses showed unilateral cryptorchidism in two fetuses from two different litters of the treated group. The identification of the same stock male being mated with these two females gave rise to the hypothesis that the male parent could genetically transmit the finding. In addition, considering also the high presence of displaced testes in the control group, the findings described were considered evidence of spontaneous pathology, often seen in this species under the experimental conditions.

Thus, based on the lack of adverse effects in this study, the NOAEL for maternal toxicity and developmental toxicity for rats was considered to be 1000 mg/kg bw/day.

 

Fatty acids, C6-12, ester with propylene glycol was administered to groups of female rats at dose levels of 500, 1500 and 2500 mg/kg bw/day. No mortality occurred in maternal animals during the study period. In the mid- and high-dose group, test article-related salivation was noted in 8 and 6 females, respectively. Further findings noted in the treated groups included hair loss, scabbing and red material on various body surfaces as well as rales and soft stool. These findings occurred infrequently and were not dose-related. Mean maternal body weights, body weight gains, net body weights, net body weight gains and gravid uterine weights were unaffected by test article administration. A slightly lower food consumption in the high-dose group was not considered to be adverse based on the lack of an effect on mean body weights. Post-implantation loss, live litter size, mean fetal body weights, fetal sex ratios, mean numbers of corpora lutea and implantation sites and the mean litter proportions of pre-implantation loss were similar across all groups. There were no external developmental variations for any fetuses and no visceral and no skeletal malformations were noted for fetuses in this study which were considered to be test substance-related. Fetal malformations and developmental variations, when observed in treatment groups, occurred infrequently or at a frequency similar to that in the control group and did not occur in a dose-related manner or were within the historical control data ranges.

Thus, no adverse effects in the maternal animals and the offspring were observed and a NOAEL of 1000 mg/kg bw/day for maternal and developmental toxicity was considered.

 

Conclusion for developmental toxicity

Four studies investigating the developmental toxicity are available within the Glycol Ester category. The studies from the category members Fatty acids, C16-18, esters with ethylene glycol (CAS 91031-31-1), Decanoic acid, mixed diesters with octanoic acid and propylene (CAS 68583-51-7), C8-10, 1,3-Butandiolester (CAS 853947-59-8) and Fatty acids, C6-12, ester with propylene glycol (CAS 85883-73-4) did not show treatment-related effects up to the highest tested dose level. Thus, no hazard for developmental toxicity was identified.

References

Agency for Toxic Substances and Disease Registry (ATSDR) (1997): Toxicological Profile for Propylene Glycol. US Department of Health and Human Services. Atlanta, US.

Agency for Toxic Substances and Disease Registry (ATSDR) (2010): Toxicological Profile for Ethylene Glycol. US Department of Health and Human Services. Atlanta, US.

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Justification for selection of Effect on developmental toxicity: via oral route:
Hazard assessment is based on the weight of evidence from all available studies.

Justification for classification or non-classification

According to Article 13 of Regulation (EC) No. 1907/2006 "General Requirements for Generation of Information on Intrinsic Properties of substances", information on intrinsic properties of substances may be generated by means other than tests e.g. from information from structurally related substances (grouping or read-across), provided that conditions set out in Annex XI are met. Annex XI, "General rules for adaptation of this standard testing regime set out in Annexes VII to X” states that “substances whose physicochemical, toxicological and ecotoxicological properties are likely to be similar or follow a regular pattern as a result of structural similarity may be considered as a group, or ‘category’ of substances. This avoids the need to test every substance for every endpoint". Since the group concept is applied to the members of the Glycol Ester Category, data will be generated from representative reference substance(s) within the category to avoid unnecessary animal testing. Additionally, once the group concept is applied, substances will be classified and labeled on this basis.

Therefore, based on the group concept, all available data on toxicity to reproduction do not meet the classification criteria according to Regulation (EC) 1272/2008 or Directive 67/548/EEC, and are therefore conclusive but not sufficient for classification.

Additional information