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Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
14 June 2019 - 23 December 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
This study was performed for the notification in other regions where authorities do not accept read-across data.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019
Report date:
2019

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
2001
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
d.d. 03 August 2018
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Dihexyl ether
EC Number:
203-987-8
EC Name:
Dihexyl ether
Cas Number:
112-58-3
Molecular formula:
C12H26O
IUPAC Name:
1-(hexyloxy)hexane
Test material form:
liquid

Test animals

Species:
rat
Strain:
Wistar
Remarks:
CRL:WI(Han)
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Sulzfeld, Germany
- Age at study initiation: at leats 13 weeks old at mating
- Weight at study initiation: 194-245 g
- Fasting period before study: No
- Housing: Successfully mated animals were housed individually in polycarbonate cages. Deep wood sawdust was use as bedding to allow digging and other normal rodent activities. Nest building material was also added into the cages.
- Diet: ad libitum, ssniff® SM R/M
- Water: ad libitum, tap water
- Acclimation period: At least 26 days

The diet and drinking water were routinely analysed and are considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study.
The bedding and nest building material were considered not to contain any contaminants that could reasobably be expected to affect the purpose or integrity of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.1-26.9 (target: 22 ± 3)
- Humidity (%): 32-69 (target: 30-70)
- Air changes (per hr): 15-20
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 18 June 2019 To: 18 July 2019

Administration / exposure

Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on exposure:
DIET PREPARATION
The test item was incorporated into ssniff® SM R/M-Z+H, to generate the test concentrations required. Based on previous experimental work with the test item, the prepared diets contained approx. 20% less test item than the nominal concentrations (the added amount of test item per kg diet). Partial evaporation of the test item during the pelleting process was considered to be the reason for this.
Therefore, the concentrations ordered from ssniff® had a correction factor of 1.25.
The pelleting process was considered not to induce an "unmixing" of the diets or particle segregation, and would prevent the potential settling out of the fine-heavy particles that could occur in handling/transport of powder diets. Di-n-hexyl ether was incorporated into the diet and mixed for up to approximately 16 minutes (approximately 8 minutes for premix preparation, and approximately 8 minutes for preparation of the complete diets). Following mixing, pellets were prepared by simple compression; no binding agents, steam, external heat, any other process or substance were used that might affect the test item or the quality of the diets. Similar diet preparation procedures were used to generate control diet (0 mg Di-n-hexyl ether/kg diet).
The prepared diets were stored frozen (approx. -20 °C) in plastic bags soon after preparation, pending and during transport to the testing facility, the prepared diets were also stored frozen. Each morning, an appropriate amount of frozen diet was thawed at room temperature, then placed into the animal cages. The next day, the remaining food was replaced with freshly thawed food in the animal cages. This way, the diet was at room temperate for not more than 27 hours during the study, with the exception of one day (27 June 2019), when it was at room temperature for 30 hours.

The stability of the test item in the diet had been verified for a period of at least 11 weeks frozen (approx. -20 °C), and for 27 hours at room temperature after thawing.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analysis of the diets for homogeneity and/or concentration of test item was performed by an analytical method validated at the Test Facility, using a validated GCFID method. The Di-n-hexyl ether content was measured on two occasions from each batch (two) used in the study: once at the start of feeding and once during the last week of the treatment.
Sampling for the measurements: Five representative test-item treated diet samples of an appropriate weight were collected at each occasion during the study, from five different locations of one representative diet container from each concentration. Additionally, at least one sample was similarly taken from the control diet, for analysis of test item concentration (i.e. a total of 16 samples were measured per occasion).

Acceptance criteria of the concentration analysis were set according to the analytical method validation, at 100 ± 20% of the expected concentrations. Acceptance criteria of the homogeneity was that the CV of replicates (from 5 different locations) should be less than 10%.
Details on mating procedure:
- Impregnation procedure: cohoused
The oestrus cycle of female animals was examined shortly before start of pairing. After acclimation, the females were paired according to their oestrus cycle with males in the morning for approximately 2-3 hours (1 male : 1 female) until at least 24 sperm positive females/group were attained. After the daily mating period, a vaginal smear was prepared and stained with 1% aqueous methylene blue solution. The smear was examined with a light microscope; the presence of a vaginal plug or sperm in the vaginal smear was considered as evidence of copulation (gestation day 0, GD 0). Sperm positive females were separated and caged individually.
Duration of treatment / exposure:
gestation day (GD)6 to GD20
Frequency of treatment:
continuously through feed
Duration of test:
GD0 tot GD20
Doses / concentrationsopen allclose all
Dose / conc.:
100 ppm
Remarks:
target concentration in diet; Mean test item intake 121.6 mg/kg bw/day
Dose / conc.:
300 ppm
Remarks:
target concentration in diet; Mean test item intake 438.9 mg/kg bw/day
Dose / conc.:
15 000 ppm
Remarks:
target concentration in diet; Mean test item intake 1210.1 mg/kg bw/day
No. of animals per sex per dose:
25
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale:
The dose levels were set by the Sponsor based on the available information of the chemical nature, characteristics of the test item, and the results of a dose range finding (DRF) study (See also any other information on materials and methods).

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at the onset of treatment (GD6) and weekly thereafter. On GD 13/14 the sperm positive females were examined for the presence of vaginal bleeding or "placental sign" (intrauterine extravasation of blood as an early sign of pregnancy in rat, which is considered to confirm implantation)

BODY WEIGHT: Yes
- Time schedule for examinations: GD 0, 3, 6, 8, 10, 12, 14, 16, 18 and 20.

FOOD CONSUMPTION AND COMPOUND INTAKE: 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: No

WATER CONSUMPTION: No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organs examined: viscera, ovaries, uterus
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
Fetal examinations:
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: Yes: half per litter
Statistics:
The statistical evaluation of data was performed with the program package SAS v9.2 or SPSS PC+4.0, by an appropriate statistical method.
Decision tree for statistical evaluation of numeric data with SAS v9.2 software (within the validated Provantis system). The normality and heterogeneity of variance between groups were checked by Shapiro-Wilk and Levene tests using the most appropriate data format (log-transformed when justified).
- No significant heterogeneity: an Anova / Ancova (one-way analysis of variance) test was carried out.
- Significant heterogeneity: Dunnett’s (Multiple Range) test was used to assess the significance of inter-group differences; identifying differences of <0.05 or <0.01 as appropriate.
This parametric analysis was the better option when the normality and heterogeneity assumptions implicit in the tests were adequate.

If either of the Shapiro-Wilk or Levene tests showed significance on the data, then a non-parametric analysis was used. A Kruskal-Wallis analysis of variance was used after Rank Transformation. If there was a positive result, the inter-group comparisons were performed using Dunn test; identifying differences of <0.05 or <0.01 as appropriate. For the SPSS PC+4.0 program package, the heterogeneity of variance between groups was checked by Bartlett's test.
- No significant heterogeneity: a one-way analysis of variance (ANOVA) was carried out.
- Significant heterogeneity: Duncan's Multiple Range test was used to assess the significance of inter-group differences.
Where significant heterogeneity was found, the normal distribution of data was examined by Kolmogorow-Smirnow test. In the case of not normal distribution, the non-parametric method of Kruskal-Wallis One-Way analysis of variance was applied. If a positive result was detected, the inter-group comparisons were performed using Mann-Whitney U-test. The Chi squared test was used for comparing group incidences against the controls.
Indices:
- Preimplantation loss: (Number of corpora lutea-Number of implantations)/(Number of corpora lutea) x100
- Postimplantation loss: (Number of implantations-Number of live foetuses)/(Number of implantations) x100
- Sex distribution: (Number of male (female) foetuses/Number of foetuses) x100
- External abnormalities/litter: (Number of foetuses with abnormality/Number of foetuses) x100
- Visceral abnormalities/litter: (Number of foetuses with abnormality/Number of foetuses) x100
- Skeletal abnormalities/litter: (Number of foetuses with abnormality/Number of foetuses) x100

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Piloerection was observed in 9 out of 24 Mid dose group animals, and 15 out of 25 High dose animals.
Considering the general, transient, and mild nature of these findings, they are considered to be treatment-related changes, not sufficient to indicate a clear adverse effect.
Thin fur on some animals was observed in the Low (1 out of 25 animals), and High dose group (1 out of 25 animals). These changes were most probably incidental, and not considered to be test item related effects.
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Test item related effect was observed on the body weight parameters and calculated body weight parameters in the High dose group. The corrected body weight and body weight gain on GD20 was 250.5 g and 26.8 g in the high dose group versus 265.5 g and 41.3 g in controls). This was considered to be a test item related effect.
No test item related effect on the body weight was observed in the test item treated Low and Mid dose groups when compared to the Control. Similarly, for these groups, no statistically significant or biologically relevant differences were seen in the body weight gain or corrected body weight gain values during the treatment period (GD 6-20) or entire study (GD 0-20) compared to the control value.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Reduced food consumption, reaching statistical significance at the onset of treatment (Day 6-8), and during the treatment (Day 10-14, Day 18-20), resulting statistically significant food consumption reduction for the treatment and the whole study period was observed in the High dose group.
No test item related effect on the food consumption was observed in the test item treated Low and Mid groups when compared to the Control.
The test item intake values (mg/kg bw/day) were calculated from the individual body weight, mean food intake and nominal diet concentration. The mean achieved dose levels were 1210.1, 438.9 and 121.6 mg/kg bw/day in the High (15000 ppm), Mid (5000 ppm) and Low (1500 ppm) dose groups, respectively.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no findings note at necropsy that were considered to be related to the treatment.
In one High dose female, a single red mass was observed on the lateral lobe of the liver. This finding was considered to be incidental, and not related to the test item. Correlated to the clinical observations in the in life phase, thin fur was observed in one animal, however, was incidental and not related to the test item.

Maternal developmental toxicity

Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
439 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
body weight and weight gain

Maternal abnormalities

Abnormalities:
no effects observed

Results (fetuses)

Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
The mean foetal weight per litter was significantly lower in the High dose group when compared to the control. Similarly, the number of foetuses with retarded body weight were significantly higher in this group. This body weight reduction is considered to be a secondary effect due to the body weight changes observed in the dams. The pattern of foetal weight and retarded weights seen in this study is consistent with other studies where maternal body weight was lower than controls; there was no evidence of a direct effect of test item on the foetuses.
The mean foetal weight per litter in the test item treated Low and Mid groups did not differ significantly from the control mean value.
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
External malformations:
no effects observed
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Based on the skeletal findings the number of malformed / variant / intact foetuses were comparable with the controls in the Low, Mid and High dose groups.
Malformations such as Lumbar-Sacral Transverse process, fused, Pelvic girdle, malpositioned in Control (1/118 foetus), Low (2/117 foetuses in 2 litters) and Mid (1/122 foetus) dose groups and Multiple Malformed Vertebrae (centrum, absent; arches, fused; misshapen; split) in the Low dose group (1/117 (2521/3) foetus) were observed.
These finding occur and correspond with the current historical control and study control data or have isolated occurrence that rather considered incidental, ascribed to individual variability than related to treatment.
Visceral malformations:
no effects observed
Description (incidence and severity):
For the visceral findings (variations), the foetal or litter based incidence in the test item treated groups was comparable with the study control and/or historical control values. The variations observed were considered as incidental, and not related to the test item treatment.

Effect levels (fetuses)

open allclose all
Key result
Dose descriptor:
NOAEL
Remarks:
foetotoxicity
Effect level:
439 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
fetal/pup body weight changes
Dose descriptor:
NOEL
Remarks:
embyotoxicity
Effect level:
1 210 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Remarks on result:
other: Based on the lack of any test-item related intrauterine effect in any treatment group.
Dose descriptor:
NOEL
Remarks:
teratogenecity
Effect level:
1 210 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Remarks on result:
other: Based on the lack of any developmental effects in any treatment group.

Fetal abnormalities

Abnormalities:
no effects observed

Overall developmental toxicity

Key result
Developmental effects observed:
no

Any other information on results incl. tables

Test concentrations and test item intake

 

Group number

Group designation

Nominal concentration in diet (ppm)

Target concentration in diet (ppm)

Target dose levels

(mg/kg bw/day)

Average measured concentrations

occasion 1

Average measured concentrations occasion 2

Mean test item intake (mg/kg bw/day)

1

Control

0

0

0

**

0

-

2

Low

1875

1500

120

1447

1463

121.6

3

Mid

6250

5000

400

5145

5284

438.9

4

High

18750

15000

1100

15566

16046

1210.1

 

**Note: Interference in one of the two blank samples is not detected and the other is less than 20% of the lowest concentration of the calibration curve.

Dose range finding study

Three groups of Hannover Wistar rats received Di-n-hexyl ether administered in ssniff® SM R/M-Z+H diet, pelleted by simple compression from Gestation Day 6 (GD6) up to

and including GD19 (sperm positive day = day 0 of pregnancy, GD0). Similarly, the control group received the control diet. Caesarean sections, necropsy of dams and examination of uterine contents were performed on GD20.

The dose levels were set by the Sponsor in agreement with the Study Director based on the available data provided by the sponsor, including the results of a 28-days DRF study,

with the aim of inducing toxic effects but no death or suffering at the highest dose(15000 ppm, approximately 1000 mg/kg bw/day).

Parameters monitored during the study included mortality and clinical observations, body weight, body weight gain and individual food consumption. Gross macroscopic examination was performed at necropsy. Maternal reproductive parameters associated with uterine examination were evaluated, and the foetuses were weighed and examined

for external abnormalities. Placentae were examined macroscopically.

Twenty-four sperm positive females were included in the study, at least six in each group. The number of confirmed pregnant dams was 6, 5, 5 and 6 in the control, Low, Mid and High dose groups, respectively.

Results

No animal was found dead during the study.

No test item-related clinical signs were observed in the study.

Body weight loss, not reaching statistically significance, was observed at the onset of treatment in the High dose group. Lower food consumption was observed in all test item-treated groups, reaching statistical significance in the High dose group. Consequently, significantly lower body weight gain, corrected body weight gain, and net body gain parameters were recorded for the High dose group animals. These changes were considered to be the results of a diet avoidance behaviour, and not a primary test item related toxic effect. No test item related macroscopic findings were present at necropsy in any of the animals in the study.

There was no statistically significant difference in foetal death in any test item treated groups compared to the control. The mean number of corpora lutea was comparable with the control in all test item treated groups. No significant differences were noted in preimplantation loss or number of implantations of the test item treated groups when compared to the control.

The early and the late embryonic loss values of the test item treated groups, and the number of dead foetuses were comparable with control. There was no statistically significant difference in the postimplantation loss or total intrauterine mortality between the test item treated and control groups.

No toxicologically relevant adverse effect of the test item was observed on the foetal parameters, including mean foetal weight, number of retarded foetuses (runts), and in the sex distribution in the Top dose group. Placentas were normal for all observed animals. There was no test item related effect on the external development of foetuses in the

study.

In conclusion, Di-n-hexyl ether, when administered in diet to pregnant Hannover Wistar rats from gestation days GD6 to GD19 up to approximately 1240 mg/kg bw/day actual concentration, was not associated with maternal toxicity, embryotoxicity, foetotoxicity or any teratogenicity effect. Reduced body weight gain parameters associated with reduced food intake, were observed in all the test item-treated groups, reaching statistical significance in the High dose group, indicating a diet avoidance behaviour, and not considered to be a primary test item-related toxic effect. No other significant effects of treatment were observed.

In selecting dose levels for the following OECD 414 study, dietary concentrations which are expected to be approximately 100, 300 and 1000 mg/kg bw/day actual dose levels are suggested (nominal 1875, 6250 and 18750 ppm).

DOSE FORMULATION ANALYSIS

The mean concentration of all measured formulations were found to be in the range of 77-86% of their nominal concentrations (1875, 6250, and 18750 ppm) and were found to be homogenous. ABased on previous experimental work with the test item, approximately 20% loss due to the diet preparation was anticipated.

No test item was detected in the control diet.

All formulations proved to be homogeneous, the relative standard deviation (RSD%) of the replicates was less than 10% (actual between 1.9 and 2.6).

Applicant's summary and conclusion

Conclusions:
Di-n-hexyl ether, when administered daily through diet to pregnant Hannover Wistar rats from gestation days GD6 to GD20 at dose levels up to 1210 mg/kg bw/day did not induce embryotoxicity, foetotoxicity, or teratogenicity. The observed body weight changes in the dams indicates a slight maternal toxicity at the dose level of 1210 mg/kg bw/day, with consequent foetal body weight differences. There were no malformations or developmental effects attributed to the test item at any dose level. The NOAEL for maternal toxicity and foetotoxicity was 439 mg/kg bw/day.
Executive summary:

A developmental dietary toxicity study was performed to assess the effects of the test item dihexyl ether on the embryonic and foetal development (including the organogenesis period) of Wistar rats in their first pregnancy. Three groups of Wistar rats received Di-n-hexyl ether administered in diet, from gestation day 6 (GD 6) up to and including gestation day 20 (GD 20). Based on the results of a dose range finding, the following doses were selected: 1500, 5000 and 15000 ppm.

No test item related mortality was observed in the study. Piloerection was observed in 9 out of 24 Mid dose group animals, and 15 out of 25 High dose animals. Considering the general, transient, and mild nature of these findings, they are considered to be treatment-related changes, not sufficient to indicate a clear adverse effect. Test item related effect was observed on the body weight parameters and calculated body weight parameters in the High dose group. This was considered to be a test item related effect. No test item related effect on the body weight was observed in the test item treated Low and Mid dose groups when compared to the Control. At necropsy of the dams, no remarkable external observations related to test item treatment were recorded for any pregnant animals. No remarkable abnormalities were observed on the placentas in any group. Due to the observed body weight changes in the dams, a slight maternal toxicity at the dose level of 1210 mg/kg bw/day is indicated.

There was no test item related effect on the intrauterine mortality parameters. The mean number of viable foetuses was comparable with the control mean in all test item treated groups. There was no toxicologically significant difference in the sex distribution of fetuses between the control and treatment groups. The mean foetal weight per litter was significantly lower in the High dose group when compared to the control. Similarly, the number of foetuses with retarded body weight, and the number of affected litters were higher in this group. This body weight reduction is considered to be a secondary effect due to the body weight changes observed in the dams. The pattern of foetal weight and retarded weights seen in this study is consistent with other studies where maternal body weight was lower than controls; there was no evidence of a direct effect of test item on the foetuses. The mean foetal weight per litter values and the number of retarded foetuses (runts) in the Low and Mid dose groups were comparable with the control. There was no test item related effects on external, visceral, or skeletal development of foetuses in the study. Foetal malformations observed in the study were considered to be incidental. The malformations showed no dose dependency, and were not regarded as a test item related effect.

In conclusion, Di-n-hexyl ether, when administered daily through diet to pregnant rats from gestation days GD6 to GD20 at dose levels up to 1210 mg/kg bw/day did not induce embryotoxicity, foetotoxicity, or teratogenicity. The observed body weight changes in the dams indicates a slight maternal toxicity at the dose level of 1210 mg/kg bw/day, with consequent foetal body weight differences. There were no malformations or developmental effects attributed to the test item at any dose level.

The following no-observed-adverse-effect (NOAEL) levels were derived:

NOAELmaternal toxicity: 439 mg/kg bw/day

Based on the lower body weight indicating maternal toxicity effect in the High dose group.

NOELembryotoxicity: 1210 mg/kg bw/day

Based on the lack of any test-item related intrauterine effect in any treatment group.

NOAELfoetotoxicity: 439 mg/kg bw/day

Based on the lack of any test-item related foetotoxicity effect in any treatment group other than reduced weight secondary to maternal body weight in the High dose.

NOELteratogenecity: 1210 mg/kg bw/day

Based on the lack of any developmental effects in any treatment group.