Isotridecan-1-ol

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

OECD 422: NOAEL = 2000 mg/kg (Hansen 1992)

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

see justification attached to chapter 13

Reason / purpose for cross-reference:

read-across source

Dose descriptor:

NOAEL

Effect level:

>= 2 000 mg/kg bw/day

Sex:

male/female

Basis for effect level:

other: no effect observed

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

>= 2 000 mg/kg bw/day

Sex:

male/female

Basis for effect level:

other: no effect observed

Reproductive effects observed:

no

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

2 000 mg/kg bw/day

Species:

rat

Additional information

There were no data concerning fertility of iso-Tridecan-1-ol available. Data were adopted by read-across from 1-Dodecanol (CAS 112-53-8). Due to structural similarities, the same results can be expected for iso-Tridecan-1-ol.

A one-generation GLP-study (reliable with restrictions) in rats (Hansen, E., 1992) was performed with the substance 1-dodecanol (CAS 112-53-8; 99%) using the Combined Repeated Dose and Reproductive/Developmental Toxicity Screening Test protocol (OECD 422). Male and female rats were administered 1-dodecanol orally via the feed at doses of 100, 500 and 2000 mg/kg/day for a period of 14 days. No effects were seen on reproductive parameters up to doses of 2000 mg/kg/day. 1-Dodecanol at the dose administered had no influence on body weight, weight gain, food consumption and reproductive efficiency in the parental generation. Pregnancy rates were not statistically altered and there were no differences in the lengths of the gestation periods. No organ toxicity was observed in the females, and there was no effect on the number of pups per litter, weight, sex ratio, or mortality rate from day 1 to 5 after birth. Therefore a NOAEL of 2000 mg/kg bw was determined both for the parental and offspring generation (male and female animals).

Effects on developmental toxicity

Description of key information

- rat, OECD 414: NOAEL(dev.tox.) = 750 mg/kg bw; NOAEL(maternal toxicity) = 250 mg/kg bw (BASF, 2003)

- rabbit, OECD 414: NOAEL (dev.tox.) = 250 mg/kg bw; NOAEL (maternal toxicity) = 75 mg/kg bw (BASF, 2020)

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

no

Principles of method if other than guideline:

OECD Guidelines for Testing of Chemicals; Proposal for updating Guideline 414: Prenatal Developmental Toxicity Study (January 22, 2001)

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Germany
- Age at study initiation: The animals were supplied at an age of about 70 - 84 days.
- Weight at study initiation: Based on the pregnant animals the body weight on day 0 varied between 146.2 - 188.1 g.
- Housing: single housing
- Diet: ground Kliba maintenance diet rat/mouse/hamster meal, supplied by PROVIMI KLIBA SA, Kaiseraugst, Switzerland; food was available to the animals ad libitum throughout the study (from the day of supply to the day of necropsy);
- Water: drinking water of tap water quality from water bottles; ad libitum
- Acclimation period: Between start of the study (beginning of the experimental phase) and first administration (day 6 p.c.) the animals were acclimated to the laboratory conditions.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24
- Humidity (%): 30 - 70
- Photoperiod (hrs dark / hrs light): 12 / 12

Route of administration:

oral: gavage

Vehicle:

other: olive oil Ph. Eur./DAB

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:

The oily test substance solutions were prepared at the beginning of the administration period and thereafter at intervals, which took into account the analytical results of the stability verification. For the preparation of the solutions, an appropriate amount of the test substance was weighed in a graduated measuring flask depending on the dose group, topped up with olive oil Ph.Eur./DAB and subsequently thoroughly mixed.

Administered standard dose volume of 5 ml/kg body weight

VEHICLE
- Concentration in vehicle: 1.2, 5 or 15 g/100 ml

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The test substance solutions were analyzed by GC. The results of the analyses of test substance solutions in olive oil Ph.Eur./DAB confirmed the correctness of the prepared concentrations. The analytical values of the samples corresponded to the expected values within the limits of the analytical method, i.e. were always above 90% and below 110% of the nominal concentrations.

Details on mating procedure:

The animals were mated by the breeder ("time- mated") and supplied on day 0 post coitum (p.c.). The animals arrived on the same day (i.e. day 0 p.c.) at the experimental laboratory. The following day was designed "day 1" p.c..

- Proof of pregnancy: vaginal plug / sperm referred to as day 0 p.c.

Duration of treatment / exposure:

day 6 through day 19 of gestation (from implantation to one day prior to the expected day of parturition)

Frequency of treatment:

1x/d

Duration of test:

on day 6 through day 19 post coitum (p.c.)

Remarks:

Doses / Concentrations:
60, 250, 750 mg/kg bw

No. of animals per sex per dose:

25 mated female Wistar rats/group

Control animals:

yes, concurrent vehicle

Maternal examinations:

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: A check was made twice a day on working days or once a day (Saturday, Sunday or on public holidays) (days 0 - 20 p.c.). The animals were examined for clinical symptoms at least once a day, or more often when clinical signs of toxicity were elicited (days 0 - 20 p .c.).

BODY WEIGHT: Yes
- Time schedule for examinations: All animals were weighed on days 0, 1, 3, 6, 8,10,13, 15, 17, 19 and 20 p.c.. The body weight change of the animals was calculated from these results. Furthermore, the corrected body weight gain was calculated after terminal sacrifice (terminal body weight on day 20 p .c. minus weight of the unopened uterus minus body weight on day 6 p .c.).

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes (With the exception of day 0, the consumption of food was determined on the same days as was body weight.)
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes / No / No data
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes / No / No data

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

OTHER:

Clinical Pathology:
Blood was taken from the retroorbital venous plexus in the morning from fasted animals. The animals were anaesthetized using isoflurane as anesthesia. The blood sampling procedure and the subsequent analysis of the blood and serum samples were carried out in a randomized sequence. The following examinations were carried out in all female animals per test group:

Hematology:
The following parameters were determined in blood with EDTA-K3 as anticoagulant using a particle counter (ADVIA 120 model; Bayer, Fernwald, Germany): leukocytes, erythrocytes, hemoglobin, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, platelets, differential blood count, reticulocytes.

Furthermore, differential blood smears were prepared and stained without being evaluated.

The clotting analyses were carried out and the prothrombin time (Hepator Quick's test) was determined.

Clinical chemistry:
An automatic analyzer was used to examine the clinicochemical parameters. The following parameters were determined: alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, serum-gamma-gIutamyltransferase, sodium, potassium, chloride, inorganic phosphate, calcium, urea, creatinine, glucose, total bilirubin, total protein, albumin, globulins, triglycerides, cholesterol, magnesium

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: dead fetuses

Fetal examinations:

- External examinations: Yes [all per litter]
- Soft tissue examinations: Yes: [half per litter]
- Skeletal examinations: Yes: [half per litter]
- Head examinations: Yes: No data

Statistics:

STATISTICS
Statistics of clinical, necropsy and fetal examinations:
Simultaneous comparison of all dose groups with the control group using the DUNNETT-test (two sided) for the hypothesis of equal means: Food consumption, body weight, body weight change, corrected body weight gain (net maternal body weight change), carcass weight, weight of unopened uterus, number corpora lutea, number of implantations, number of resorptions, number of live fetuses, proportions of preimplantation loss, proportions of postimplantation loss, proportions of resorptions, proportion of live fetuses in each litter, litter mean fetal body weight, litter mean placental weight.
Organ weights (liver, kidneys, spleen): Non-parametric one way analysis using KRUSKAL-WALLIS- test (two-sided). If the resulting p-value was equal or less than 0.05, a pairwise comparison of each dose group with the control group was performed using the WILCOXON-test (two-sided) for the equal medians.
Pairwise comparison of each dose group with the control group using Fishers exact test (one-sided) for the hypothesis of equal proportions: Female mortality, females pregnant at terminal sacrifice, number of litters with fetal findings;
Pairwise comparison of each dose group with the control group using the Wilcoxon-test (one-sided) for the hypothesis of equal medians: Propotions of fetuses with malformations, variations and/or unclassified observations in each litter.

Statistics of clinical pathology:
Means and standard deviations of each test group were calculated for several parameters.
Clinical pathology except reticulocytes and differential blood count. Non-parametric one way analysis using KRUSKAL-WALLIS test (two sided). If the resulting p-value was equal or less than 0.05, a pair wise comparison of each dose group with the control group was performed using Wilcoxon-te t (two-sided) for the equal medians.

Details on maternal toxic effects:

Maternal toxic effects:yes. Remark: (at 750 mg/kg body weight/day)

Details on maternal toxic effects:
Mortality:
There were no substance-related or spontaneous mortalities in any of the groups.

Clinical symptoms:
All high dose (750 mg/kg body weight/day) and the majority (17 out of 25) of the mid dose (250 mg/kg body weight/day) animals showed transient salivation immediately after treatment on one or several days of the treatment period; however, the observed salivation persisted in the respective females only for a few minutes after the actual gavaging had taken place. This substance-induced symptom was observed first for high dose females Nos. 98, 99 and 100 on day 7 p.c. and for mid dose rat No. 75 on day 12 p.c.. After cessation of treatment on day 19 p.c., salivation did not occur any longer.
The observed temporary salivation of the animals is considered to be substance-induced. It is very likely, that this finding was induced by bad taste of the test substance or local affection of the upper digestive tract. Salivation itself is not assessed as an adverse or toxic effect.

Additionally, 4 high dose dams (Nos. 77, 83, 98 and 100) showed urine-smeared fur on gestation days 10 - 13 or 17 - 20. This clinical finding is a sign of discomfort of the rats and is probably substance-induced.

No indications for disturbances of the general behavior, however, occurred in the dams of test groups 0 and 1 (0 and 60 mg/kg body weight/day).

Food consumption:
The mean food consumption of the high dose dams (750 mg/kg body weight/day) was statistically significantly reduced (up to about 11 % below the concurrent control value) on treatment days 6 - 10 p.c.. Thereafter, food consumption of the top dose rats was similar to control values and was not influenced in a dose-related manner.
The food consumption of the females of test groups 1 and 2 (60 and 250 mg/kg body weight/day) was unaffected and did not show any statistically significant or biologically relevant differences in comparison to the controls.
The transient reduction in food consumption of the high dose dams at initiation of dosing is considered to be substance-induced; however, the decreased food intake had no corroborative effects on body weight data and corrected body weight gain of these females.

Body weight data:
There were no statistically significant or biologically relevant differences between the controls and the substance-treated dams in terms of body weights and body weight gain during the study. The observed slight and transient, but statistically significant reductions in food consumption at 750 mg/kg on the first treatment days induced no adverse effects on body weight development of these dams.

Corrected body weight gain (net maternal body weight change):
The corrected body weight gains (terminal body weight on day 20 p.c. minus weight of the unopened uterus minus body weight on day 6 p.c.) of the dams of test groups 1 - 3 (60; 250 and 750 mg/kg body weight/day) revealed no differences of any biological relevance to the corresponding control group. Moreover, a clear relation to dosing was not present.

Examinations of the dams at termination:
--------------------------------------
Hematology:
There were no treatment- related changes in the hematological parameters measured.

Clinical chemistry:
At the end of the administration period serum enzyme examinations revealed slightly, but statistically significantly increased alanine aminotransferase activities in the high dose animals. The other serum enzymes were not affected by the test compound.
Blood chemistry investigations showed decreased total protein and globulin concentrations and increased triglyceride levels in the serum of the high dose females. No treatment- related changes were observed in the other blood chemistry parameters.
The slight changes seen in the clinical chemistry parameters alanine aminotransferase, total protein, globulins and triglycerides of the high dose animals are considered to be test substance-related and are indicative for a mild adverse effect on the liver.

Other deviations:
There are additional statistically significant intergroup differences in the results of clinical pathology testing. These deviations are marginal, incidental, or lack dose-response relationship. Accordingly, these findings are considered to be of no toxicological significance.

Uterus weight:
The mean gravid uterus weights of the animals of test groups 1 - 3 (60, 250 or 750 mg/kg body weight/day) were not influenced by the administration of the test substance. The differences between these groups and the control group revealed no dose-dependency and were assessed to be without biological relevance.

Liver weight:
Absolute and relative mean liver weights were statistically significantly increased at the high dose group (750 mg/kg body weight/day) and were about 14% (absolute) or 18% (relative) above control values. The increased liver weights at the top dose are in line with the slight changes seen in different clinical chemistry parameters (i.e. alanine aminotransferase, total protein, globulins and triglycerides); they are considered as substance-induced.
Absolute and relative liver weights of the dams of test groups 1 and 2 (60 and 250 mg/kg body weight/day) were similar to the respective control values and did not show any toxicologically significant changes.

Kidney weights:
Absolute and relative mean kidney weights of the dams of test groups 1, 2, and 3 (60, 250 and 750 mg/kg body weight/day) were similar to the respective control values and did not show any toxicologically significant differences.

Spleen weight:
Absolute mean spleen weights were statistically significantly decreased at the high dose group (750 mg/kg body weight/day) and were about 9% lower than the control values. As the more important relative spleen weights of the top dose dams remained unaffected, this is considered to be spontaneous in nature and not as substance-induced .
The absolute and relative spleen weights of the low and mid dose rats (60 and 250 mg/kg body weight/day) were similar to the respective control values and did not show any toxicologically significant changes.

Necropsy findings:
There were no substance-related observations at necropsy in any of the dams.
Control dam (No. 10) had a hemorrhagic thymus. This gross finding is considered to be spontaneous in nature and is probably related to the method how the rats were killed.

Reproduction data of dams:
--------------------------
The conception rate reached 100% in test groups 0 and 1 (0 and 60 mg/kg body weight/day), 92% in test group 2 (250 mg/kg body weight/day) and 96% in test group 3 (750 mg/kg body weight/day). As all rats that became pregnant had implantation sites at necropsy, a sufficient number of females for the purpose of the study were available.
There were no substance-related and/or biologically relevant differences between the different test groups in conception rate, in the mean number of corpora lutea and implantation sites or in the values calculated for the pre- and the postimplantation losses, the number of resorptions and viable fetuses.
Low dose dam No. 34 had one dead fetus, but also 9 live fetuses in the uterus. Mid dose dam No. 62 was only pregnant by stain. It had only two very early resorptions, but no viable fetuses in the uterus. The isolated occurrence of one low dose rat with one dead fetus and of one mid dose female with total resorptions does not suggest any relation to treatment. Moreover, both findings occur also occasionally as spontaneous findings in the strain of rats used for this study.
Thus, all differences observed in respect to the gestational parameters in the various test groups were within the normal range of deviations for animals of this strain and age.

Dose descriptor:

NOAEL

Effect level:

250 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
Examinations of the fetuses after dissection from the uterus:

Sex distribution of fetuses:
The sex distribution of the fetuses in test groups 1 - 3 (60, 250 and 750 mg/kg body weight/day) was comparable with that of the control fetuses. The observable differences in comparison to the concurrent control are without any biological relevance.

Weight of placentae:
The mean placental weights in the substance-treated groups (60, 250 and 750 mg/kg body weight/day) were similar to the corresponding control values and did not show any dose dependency.

Weight of fetuses:
The mean fetal body weights in test groups 1, 2 and 3 (60, 250 and 750 mg/kg body weight/day) were not influenced by the test substance administration and were very similar to the corresponding control values.

Soft tissue examination of the fetuses:
Soft tissue malformations were recorded for one low dose and one high dose fetus (60 and 750 mg/kg body weight/day) each.
Unilateral hydronephrosis in combination with hydroureter occurred in female fetus No. 8 from low dose dam No. 44 and a situs inversus was seen in male fetus No. 6 from high dose dam No. 76. The isolated occurrence of these soft malformations does not suggest any relation to dosing.
In total, none out of the 92 examined control fetuses (from 25 litters), one out of 104 low dose fetuses [= 1.0%] (in one out of 25 litters [= 4.0%]), none out of the examined 87 mid dose fetuses (from 22 litters) and one out of 88 high dose fetuses (in one out of 24 litters [= 4.2%]) showed soft tissue malformations. The mean percentages of affected fetuses/litter with soft tissue malformations amounted to 0.0, 0.8, 0.0 and 1.4%, respectively without attaining statistical significance in any of the test groups (60, 250 or 750 mg/kg body weight/day).
Two soft tissue variations (uni- or bilateral dilation of the renal pelvis and/or ureter) were detected in each group including the controls in 8 - 12 fetuses from 4 - 9 litters without any dose-response relationship.
The mean percentages of affected fetuses/litter with total soft tissue variations amounted to 11.8% (control), 13.1% (60 mg/kg body weight/day), 8.5% (250 mg/kg body weight/day) and 9.7% (750 mg/kg body weight/day), respectively. Thus, a relation to dosing is not present and a substance-induced effect concerning the occurrence of soft tissue variations can be excluded with certainty.

No so-called unclassified soft tissue observation (like blood imbibition of kidney(s)) was recorded in any of the fetuses.

Skeletal examination of the fetuses:
The only skeletal malformations, which occurred, were observed for low dose fetus No. 10 from dam No. 34. This fetus was already dead and showed two external malformations, i.e. gastroschisis and scoliosis, when it was developed from the uterus of its mother. The observed additional skeletal malformations (i.e. misshapen thoracic arch (with changed cartilage) and fused rib (with present cartilage)) fit well to the above mentioned external findings.
In total, none of the 105 control fetuses (from 25 litters), one of 115 low dose fetuses [= 0.9%] in one of 25 litters [= 4.0%], none of the 99 mid dose fetuses (from 22 litters) and none of the 103 high dose fetuses (from 24 litters) showed skeletal malformations. The mean percentages of affected fetuses/litter with skeletal malformations amounted to 0.0, 0.7, 0.0, and 0.0%.
The occurrence of two skeletal malformations in one low dose fetus in the absence of any further skeletal malformations in the other examined fetuses from test groups 0 - 3 (0, 60, 250 and 750 mg/kg body weight/day) does not suggest a substance - induced background but is considered to be spontaneous in nature.
In all groups signs of skeletal variations with or without involvement of corresponding cartilaginous structures elicited. The observed variations were related to the skull (supraoccipital and/or basioccipital holes; incomplete ossification of the entire skull, basisphenoid, frontal, parietal, interparietal, supraoccipital and/or hyoid), the vertebral column (incomplete, dumbbell or bipartite ossification of cervical, thoracic, lumbar and/or sacral vertebrae ; supernumerary thoracic vertebra; fused sacral centrum and arch, misshapen sacral vertebra), the ribs (supernumerary 14 th, cervical or wavy ribs), the sternum (misshapen sternebra; unilateral, incomplete, bipartite or missing ossification of sternebra) and the pelvic girdle (incomplete ossification of pubis). The mean percentages of affected fetuses/litter with skeletal variations amounted to 95.1, 93.9, 93.0, and 96.3% at 0, 60, 250 or 750 mg/kg body weight/day. Most of the noted skeletal variations appeared without a clear relation to dosing, without biologically relevant differences between the groups and/or can be found at a comparable frequency in the historical control data. This assessment includes the only skeletal variation, misshapen sacral vertebra, which occurred at a statistically significantly higher rate at 60 and 750 mg/kg body weight/day in comparison to the concurrent control group; 0.0%, 3.1 %* (* = pAdditionally, some isolated cartilage findings without any impact on the respective bony structures, which were designated as unclassified cartilage observations, occurred in all groups including the controls. The observed unclassified cartilage findings were related to the skull, the vertebral column, the ribs and the sternum. The mean percentages of affected fetuses/litter with these findings amounted to 26.9, 32.3, 34.0, and 20.7% at 0, 60, 250 or 750 mg/kg body weight/day. A toxicological relevance for these findings, which did not show any relation to dosing, can be excluded with certainty.

Dose descriptor:

NOAEL

Effect level:

750 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: embryotoxicity

Dose descriptor:

NOAEL

Effect level:

750 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: fetotoxicity

Dose descriptor:

NOAEL

Effect level:

750 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: teratogenicity

Abnormalities:

no effects observed

Developmental effects observed:

no

Executive summary:

DISCUSSION AND CONCLUSION

----------------------------------------

Tridecanol H was administered to pregnant Wistar rats daily by stomach tube from implantation to one day prior to the expected day of parturition (days 6 - 19 post coitum).

Substance-related signs of maternal toxicity occurred at 750 mg/kg body weight/day . The most obvious clinical effects on the high dose dams were transient salivation in all dams immediately after treatment on certain days, urine smeared fur in 4 dams and statistically significantly reduced food consumption at initiation of treatment period (days 6 - 10 p.c .) .

Regarding clinical pathology, statistically significantly increased alanine aminotransferase activities, decreased total protein and globulin concentrations, and increased triglyceride levels in the serum of the high dose females were recorded . These changes, which were accompanied by statistically significantly increased absolute and relative liver weights (about 14% or 18% respectively above control values), are indicative for a mild adverse effect on the liver.

The only substance-induced finding on the mid dose dams (250 mg/kg body weight/day) consisted in transitory salivation in 17 out of 25 rats, which, by itself and if seen in isolation, is not assessed as an adverse or toxic effect.

No substance-induced effects on the dams occurred at the low dose level (60 mg/kg body weight/day).

The oral administration of Tridecanol H to the dams at all 3 dose levels (60 ; 250 and 750 mg/kg body weight/day) had no influence on the gestational parameters. Conception rate, mean number of corpora lutea, total implantations, resorptions and live fetuses, fetal sex ratio or in the values calculated for the pre- and the postimplantation losses were unaffected by treatment.

No substance- related differences were recorded for placental and fetal body weights .

The external, soft tissue and/or skeletal (including cartilage) examinations of the fetuses revealed no differences between the control and the substance-treated groups, which might be related to the test substance administration . Number and type of fetal external, soft tissue and skeletal findings, which were classified as malformations and/or variations, recorded for the 60 ; 250 and 750 mg/kg fetuses were unaffected by treatment. These findings appeared without a clear relation to dosing and/or were seen at incidences previously found to occur spontaneously in control fetuses of this strain of rats . Additionally, there was not found any specific malformation pattern which could be indicative of a selective teratogenicity . Thus the test substance evoked no signs of prenatal developmental toxicity and in particular no indications for teratogenicity at dose levels up to and including 750 mg/kg body weight/day.

Based on these results, the no observed adverse effect level (NOAEL) for maternal toxicity is 250 mg/kg body weight/day, while it is 750 mg/kg body weight/day for prenatal developmental toxicity.

Reference 2

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

05-06-2019 - 20-05-2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.31 (Prenatal Developmental Toxicity Study)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH (Breeder: Charles River Laboratories, France)
- Age at study initiation: 15-17 weeks
- Weight at study initiation: Based on the pregnant animals the body weight on GD 0 varied between 3285 – 4235 g.
- Fasting period before study: no
- Housing: During the acclimatization and study period, the rabbits were housed singly in Type 4X03B700CP cages supplied by TECNIPLAST Deutschland GmbH, Hohenpeißenberg, Germany (floor space 4264 mm², internal height 450 mm). For enrichment, wooden gnawing blocks were added (Typ KNH E-041, supplied by Abedd® Lab. and Vet. Service GmbH, Vienna, Austria).
- Diet and Water (e.g. ad libitum): The food used was pelleted Kliba maintenance diet rabbit and guinea pig “GLP”, supplied by Granovit AG, Kaiseraugst, Switzerland. Food and drinking water (potable tap water in water bottles) were available ad libitum throughout the study (from the day of arrival to the day of necropsy).
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
The animals were accommodated in fully air-conditioned rooms in which central air conditioning maintained a range of temperature of 17-21°C and a range of relative humidity of 45-65%. The air exchange rate was 15 times per hour. There were no deviations from these limits during the entire study.
The day/night cycle was generally 12 hours (12 hours light from 6.00 h to 18.00 h and 12 hours darkness from 18.00 h to 6.00 h).


IN-LIFE DATES: From: To: 05 Jun 2019 to 25 Jul 2019

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Remarks:

(0.5% CMC suspension in deionized water [with 10 mg/100 mL Tween 80])

Details on exposure:

The test substance preparations were prepared at the beginning of the administration period and thereafter at intervals, which took into account the period of established stability. The preparations were kept at room temperature.

For the test substance preparation, the specific amount of the test substance was weighed, topped up with 0.5% CMC suspension in deionized water (with 10 mg/100 mL Tween 80) in a calibrated beaker and intensely mixed with a magnetic stirrer.


Before and during administration, the preparations were kept homogeneous with a magnetic stirrer.

The volume administered each day was 10 ml/kg body weight. The calculation of the administration volume was based on the most recent individual body weight.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The analytical investigations of the test substance preparations were carried out at the Analytical Chemistry Laboratory of Experimental Toxicology and Ecology of BASF SE, Ludwigshafen, Germany.

The stability of the test substance in 0.5% CMC suspension in deionized water (with 10 mg/100 mL Tween 80) over a period of a maximum of 7 days at room temperature had been verified prior to the start of the study.

Samples of the test substance preparations were sent to the analytical laboratory twice dur-ing the study period (at the beginning of administration [samples 1-9] and after the end of the in-life period [retain sample 6R]) for verification of the concentrations. The samples taken for the concentration control analyses at the beginning of administration were also used to verify the homogeneity of the samples of the low- and high-dose levels each (25 and 250 mg/kg bw/d). Three samples (two from the top, middle and bottom) were taken for each of these preparations from the preparation vessel with a magnetic stirrer running.

All test samples, plus a duplicate set of reserve samples, were withdrawn by staff of the Reproduction Toxicology. All reserve samples and further samples were stored at the Laboratory Reproduction Toxicology frozen (at -20°C) Analysis of these samples were performed in case of equivocal analytical results with the original samples or after loss of/damage to original samples after agreement by the Study Director. Reserve samples were described by the suffix “R” in the report. Following finalization of the report, all analytical samples, including reserve samples, were discarded.


Concentration control analyses of the test substance preparations
The results of the analyses of the test substance preparations in 0.5% CMC suspension in deionized water (with 10 mg/100 mL Tween 80) confirmed the correctness of the prepared concentrations. The measured concentrations of the samples (mean values of the samples 3 - 5 and 7 - 9 and the single value of sample 6R) of treatment groups corresponded to the expected values within the limits of the analytical method, i.e. were above 90% and below 110% of the nominal concentrations.

Details on mating procedure:

After an acclimatization period of at least 5 days, the does were fertilized by means of artificial insemination.

A synthetic hormone (0.2 mL), which stimulates release of LH and FSH from the anterior pituitary lobe (Receptal®) was injected intramuscularly to the female rabbits about 1 hour before insemination. The ejaculate samples used for the artificial insemination were obtained from male New Zealand White rabbits of the same breed as the females. Each female was inseminated with the sperm of a defined male donor as documented in the raw data. The male donors were kept under conditions (air conditioning, diet, water) comparable to those of the females participating in this study.

Duration of treatment / exposure:

The day of insemination was designated as GD 0 (beginning of the study) and the following day as GD 1.

The test substance was administered to the animal orally by gavage from implantation to one day prior to the expected day of parturition (GD 6-28) always at approximately the same time of day. The animals of the control group were treated in the same way with the vehicle (0.5% CMC suspension in deionized water [with 10 mg/100 mL Tween 80]).

Frequency of treatment:

once daily

Dose / conc.:

25 mg/kg bw/day (nominal)

Dose / conc.:

75 mg/kg bw/day (nominal)

Dose / conc.:

250 mg/kg bw/day (nominal)

No. of animals per sex per dose:

25

Control animals:

yes, concurrent vehicle

Details on study design:

The high dose was selected based on signs of toxicity noted at dose levels of 500 and 1000 mg/kg bw/d in a previously conducted test study and in a maternal toxicity range-finding study which preceded this definitive prenatal developmental toxicity study.
In the test study 3 non-pregnant female NZW rabbits per group were gavage exposed to the test compound over a maximum of 3 weeks, at dose levels of 300 and 1000 mg/kg bw/d. The 1000 mg/kg bw/d dose caused an immediate disruption of food consumption in all females, during the first treatment days the animals consumed only about 3-14 g food per day, lost body weight and showed hypothermia, poor general condition and no/reduced defecation. Thus, they were killed moribund for humane reasons. At 300 mg/kg bw/d no relevant signs of toxicity were noted.
In the maternal toxicity range‐finding study, 5 presumed pregnant NZW rabbits were administered the test substance via oral gavage from gestational day (GD) 6 through GD 28, at doses of 150 and 500 mg/kg bw/d.
The 500 mg/kg bw/d dose caused in the pregnant rabbits an immediate disruption of food consumption, in the first 10 days of treatment consumed only about 6-27 g food per day and lost body weight. Three does aborted and one was killed in moribund condition after showing dehydration and abdominal posture. All does in this dose group had no reduced defecation. At 150 mg/kg bw/d no relevant signs of toxicity were noted. Clinical pathology, organ weights and macroscopic pathology revealed no relevant findings.
Based on the above described mortality and abortions, the dose level of 500 mg/kg bw/d was considered to be potentially lethal to the dams in the OECD 414 study.
Considering the specific characteristic of rabbit gastrointestinal physiology and the consequences of their disruption for the maintenance of pregnancy in rabbits, the selected high-dose for the present study represented half of this lethal dose. This approved procedure of decreasing a lethal dose by a factor of two to become the high dose in a subsequent regulatory study meets the principles of guidelines OECD 414 (adopted 2001) and OPPTS 870.3700 (US EPA), as well as ECHA practical guide 10 (“how to avoid unnecessary testing on animals”; chapter 4 “animal welfare”; ECHA-10-B-17-EN, 2010) which is in in compliance with EU Directive 86/609/EEC on animal protection. The oral route was selected since this has proven to be suitable for the detection of a toxicological hazard.

Maternal examinations:

Mortality
A check was made twice a day on working days or once a day on Saturdays, Sundays or on public holidays (GD 0-29).

Clinical symptoms
A cage-side examination was conducted at least once daily for any signs of morbidity, pertinent behavioral changes and signs of overt toxicity. If such signs occurred, the animals were examined several times daily (GD 0-29).
During the administration period (GD 6-28) all animals were checked daily for any abnormal clinical signs before the administration as well as within 5 hours after the administration.

Food consumption
The consumption of food was recorded daily during GD 0-29.

Body weight data
All animals were weighed on GD 0, 2, 4, 6, 9, 11, 14, 16, 19, 21, 23, 25, 28 and 29. The body weight change of the animals was calculated based on the obtained results.

Corrected (net) body weight gain
Furthermore, the corrected body weight gain was calculated after terminal sacrifice (terminal body weight on GD 29 minus weight of the unopened uterus minus body weight on GD 6).

Clinical Pathology
In the morning blood was taken from the ear vein from not-fasted animals without anesthesia. The blood sampling procedure and subsequent analysis of blood and serum samples were carried out in a randomized sequence.
The assays of blood and serum parameters were performed under internal laboratory quality control conditions with reference controls to assure reliable test results.
The results of clinical pathology examinations were expressed in International System (SI) units.

The following examinations were carried out in all animals per test group:

Hematology
The following parameters were determined in blood with EDTA K3 as anticoagulant using a particle counter (Advia 120 model; Bayer, Fernwald, Germany):

Parameters and methods:

Parameter Unit Method

Leukocyte count
(WBC) giga/L cytochemistry coupled with flow cytometry

Erythrocyte count
(RBC) tera/L flow cytometric laserlight scattering

Hemoglobin
(HGB) mmol/L cyanmethemoglobin meth-od; according to ICSH

Hematocrit
(HCT) L/L calculation:
MCV x erythrocytes
Mean corpuscular volume
(MCV) fL RBC/PLT method; mean of RBC volume distribution curve (histogram)
Mean corpuscular hemoglobin
(MCH) fmol calculation:
hemoglobin/erythrocytes

Mean corpuscular hemoglobin con-centration
(MCHC) mmol/L calculation:
hemoglobin/hematocrit

Platelet count
(PLT) giga/L flow cytometric laserlight scattering

Differential blood count % and giga/L cytochemistry coupled with flow cytometry

Reticulocytes (RETA) giga/L cytochemistry coupled with flow cytometry

 
Clinical chemistry
An automatic analyzer (Cobas c501; Roche, Mannheim, Germany) was used to examine the clinicochemical parameters

Parameters and methods:

Enzyme (systematic name and system number) Unit Method, wave-length and measuring tem-perature (LOQ)

Alanine aminotransferase
(ALT)
(L-alanine: 2-oxoglutarate aminotransferase;
EC 2.6.1.2.) µkat/L kinetic UV test, 340 nm; 37°C,
(0.08 µkat/L)
Aspartate aminotransferase
(AST)
(L-aspartate: 2-oxoglutarate aminotransferase;
EC 2.6.1.1.) µkat/L kinetic UV test, 340 nm; 37°C,
(0.08 µkat/L) J. Clin. Chem. Clin. Biochem. 8, 658-660 (1970);
Alkaline phosphatase
(ALP)
(orthophosphoric acid monoester phosphohydrolase;
EC 3.1.3.1.) µkat/L kinetic color test, 415 nm, 37°C,
(0.084 µkat/L)
-Glutamyltransferase
(GGT)
( -glutamyl) peptide: aminoacid--glutamyl-transferase;
EC 2.3.2.2.) nkat/L kinetic color test, 415 nm, 37°C,
(25 nkat/L)
Inorganic phosphate
(INP) mmol/L molybdate reaction
(0.1 mmol/L)
Calcium
(CA) mmol/L o-cresolphthalein complex without deproteinization
(0.2 mmol/L)
Urea
(UREA) mmol/L enzymatic determination with the urease/ glutamate dehydro¬genase method
(0.5 mmol/L)
Creatinine
(CREA) µmol/L enzymatic determination with the creatininase/ creatinase /sarcosinoxidase method
(5 µmol/L)
Glucose
(GLUC) mmol/L hexokinase/glucose-6-phosphate dehydrogenase method
(0.11 mmol/L)
Total bilirubin
(TBIL) µmol/L DPD method
(0.56 µmol/L)
Total protein
(TPROT) g/L biuret method
(2 g/L)
Albumin
(ALB) g/L bromocresol green method
(3.2 g/L)
Globulins
(GLOB) g/L difference between total protein and albumin

Triglycerides
(TRIG) mmol/L enzymatic color test with lipase esterase/ glycerokinase/ glycerol-3-phosphate oxidase/4-amino-phenazone
(0.1 mmol/L)
Cholesterol
(CHOL) mmol/L enzymatic determination with cholesterol esterase/ cholesterol oxidase/4-amino-phenazone (CHOD-PAP method)
(0.1 mmol/L)


Necropsy
On GD 29 all surviving does were sacrificed by an intravenous injection of pentobarbital in a randomized sequence. The sacrificed animals were necropsied and assessed by gross pathology, special attention being given to the reproductive organs.

Organ weights
The following weights were determined in all does sacrificed on schedule:
1. Adrenal glands
2. Kidneys
3. Liver
4. Spleen

All paired organs were weight together (left and right).

The carcass weights (GROSSE-System) were transferred to the ACOPAT-System to calculate the relative organ weights.

Organ/tissue fixation
The following organs or tissues were fixed in 4% neutral-buffered formaldehyde solution:

1. All gross lesions
2. Adrenal glands
3. Kidneys
4. Liver
5. Spleen
6. Target organs

No further examinations or procedures were performed in this study.

Ovaries and uterine content:

On GD 29, the surviving does were sacrificed in randomized order by intravenous injection of pentobarbital (Narcoren®; dose 2 mL/animal). After exsanguination, the animals were nec-ropsied and assessed by gross pathology.

The uteri and the ovaries were removed and the following data were recorded:
- Weight of the unopened uterus
- Number of corpora lutea
- Number and distribution of implantation sites classified as:
• Live fetuses
• Dead implantations:
a) Early resorptions (only decidual or placental tissues visible or according to SALEWSKI from uteri from apparently non-pregnant animals and the empty uterus horn in the case of single horn pregnancy)
b) Late resorptions (embryonic or fetal tissue in addition to placental tissue visible)
c) Dead fetuses (hypoxemic fetuses which did not breathe spontaneously after the uterus had been opened)

After the weight of the uterus had been determined, all subsequent evaluations of the does (except of gross pathology including organ sampling and weights) and the gestational parameters were conducted by technicians unaware of treatment group in order to minimize bias. For this purpose, animal numbers were encoded.

Fetal examinations:

All fetal analyses were conducted by technicians unaware of the treatment group, in order to minimize bias.

Examinations of the fetuses after dissection from the uterus
At necropsy each fetus was weighed and examined macroscopically for external findings. Furthermore, the viability of the fetuses and the condition of placentas, umbilical cords, fetal membranes, and fluids were examined. Individual placental weights were recorded.

Thereafter, the fetuses were sacrificed by an intraperitoneal injection of pentobarbital (Narcoren®; dose: 0.2 mL/fetus; one part Narcoren® diluted with one part physiological saline).

Soft tissue examination of the fetuses
After the fetuses had been sacrificed, the abdomen and thorax were opened in order to examine the organs in situ before they were removed. The heart and the kidneys were sectioned in order to evaluate the internal structure.

The sex of the fetuses was determined by examination of the gonads in situ.

After these examinations, the heads of approximately one half of the fetuses per doe (and the heads of any fetus which revealed severe findings during the external examination, e.g. anophthalmia, microphthalmia or hydrocephalus) were severed from the trunk. These heads were fixed in BOUIN’s solution and were, after fixation, processed and evaluated according to WILSON’s method (WILSON and WARKANY). About 10 transverse sections were prepared per head. After the examination the heads were discarded.

All fetuses (including those without heads) were skinned and fixed in ethyl alcohol. After fixation for approx. 1-5 days, the intact fetuses were removed from the fixative and a transversal incision was made into the frontal/parietal head bones. The two halves of the calvarium were cautiously bent outward and the brain was thoroughly examined. Subsequently, these fetuses were placed back into the fixative for further fixation.

Skeletal examination of the fetuses
After fixation in ethyl alcohol, the skeletons (with and without skulls) were stained according to a modified method of KIMMEL and TRAMMELL. The stained skeletons were placed on an illuminated plate, investigated and archived individually.

Evaluation criteria for assessing the fetuses
Classification and assessment of fetal findings is a matter of ongoing discussion (see e.g. BELTRAME and GIAVINI, CHAHOUD, SOLECKI). Despite considerable efforts to harmonize the nomenclature used to describe observations of fetal morphology, the terms still vary considerably between laboratories, investigators and textbooks in the fields of teratology and developmental toxicity.

In the present study the internationally harmonized glossary of WISE et al. (1997) and the updated version MAKRIS et al. (2009) was essentially used to describe findings in fetal morphology. Classification of these findings was based on the terms and definitions proposed by CHAHOUD and SOLECKI:
Malformation
A permanent structural change that is likely to adversely affect survival or health.
Variation
A change that also occurs in the fetuses of control animals and/or is unlikely to adversely affect survival or health. This includes delays in growth or morphogenesis that have otherwise followed a normal pattern of development.

The term "unclassified observation" was used for those fetal findings, which could not be classified as malformations or variations.

All fetal findings were listed in tables according to these classifications.
 

Statistics:

Food consumptiona), body weight, body weight change, corrected body weight gain (net maternal body weight change), carcass weight, weight of unopened uterus, number of corpora lutea, number of implantations, number of resorptions, number of live fetuses, proportions of preimplantation loss, proportions of postimplantation loss, proportions of resorptions, proportion of live fetuses in each litter, litter mean fetal body weight, litter mean placental weight: Simultaneous comparison of all dose groups with the control group using the DUNNETT-test (two-sided) for the hypothesis of equal means

Female mortality, females pregnant at terminal sacrifice, number of litters with fetal findings: Pairwise comparison of each dose group with the control group using FISHER'S EXACT test (one-sided) for the hypothesis of equal proportions

Proportions of fetuses with malformations, variations and/or unclassified observations in each litter: Pairwise comparison of each dose group with the control group using the WILCOXON-test (one-sided) for the hypothesis of equal medians

Blood parameters: For parameters with bidirectional changes:
Non-parametric one-way analysis using KRUSKAL-WALLIS test. If the resulting p-value was equal or less than 0.05, a pairwise comparison of each dose group with the control group was performed using WILCOXON-test (two-sided) for the hypothesis of equal medians

Weight parameters: Non-parametric one-way analysis using KRUSKAL-WALLIS test (two-sided). If the resulting p-value was equal or less than 0.05, a pair wise comparison of each dose group with the control group was per-formed using WILCOXON-test (two-sided) for the equal medians

Indices:

The conception rate (in %) was calculated according to the following formula:
number of pregnant animals / number of fertilized animals x 100

The preimplantation loss (in %) for each individual pregnant animal which underwent scheduled sacrifice was calculated according to the following formula:
number of corpora lutea – number of implantations / number of corpora lutea x 100

The postimplantation loss (in %) for each individual pregnant animal which underwent scheduled sacrifice was calculated according to the following formula:
number of implantations – number of live fetuses / number of implantations x 100

Historical control data:

yes

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

In total, reduced defecation was observed in five control, two low-dose, six mid-dose and seven high-dose females (0, 25, 75 and 250 mg/kg bw/d). No defecation was observed in two control, one low-dose, three mid-dose and three high-dose females.
There were no further clinical findings in the other does in this study.

Mortality:

no mortality observed

Description (incidence):

There were no test substance-related or spontaneous mortalities in any of the does in the study.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

No statistically significant difference was observed for the mean body weights (BW) and the mean body weight gain (BWC) of the high-dose females (250 mg/kg bw/) when compared to the concurrent control group. However, on GD 6, these does lost weight (-16.6 g vs. 28.4 g in the control), and the body weight remained below control during most of the treatment peri-od. Only towards the end of treatment the high-dose mean body weights recovered and were comparable to the control again. If calculated for the treatment period (GD 6-28), the high-dose does gained about 8% less weight in comparison to the control does (without attaining statistical significance).

The mean BW and the average BWC of the low- and mid-dose groups (25 and 75 mg/kg bw/d) were generally comparable to the concurrent control group throughout the entire study period. The statistically significantly changed body weight gain values in test group 2 on GD 23-25, as well as in test group 1 calculated for GD 6-28, are assessed as incidental and not treatment-related.

Corrected (net) body weight gain:
Mean carcass weights and corrected body weight gain (terminal body weight on GD 29 minus weight of the unopened uterus minus body weight on GD 6) were not significantly different between all test groups including controls (0, 25, 75 or 250 mg/kg bw/d).

For further details see supplementary tables IA-007-012 in the attachement.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

In comparison to the control group, the mean food consumption of the does in test group 3 (250 mg/kg bw/d) was reduced during major parts of the treatment period, attaining statistical significance on GD 6-13 and GD 16-17 (up to 31% below control). Although these animals partly compensated the lower food intake during the last treatment week, the high-dose does consumed overall 8% less food than the concurrent control does during the treatment period (GD 6-28).

The food consumption of the low- and mid-dose rabbits (25 and 75 mg/kg bw/d) was comparable to the concurrent control (0 mg/kg bw/d) throughout the entire study period. The statistically significantly increased food consumption value in test group 1 on GD 23-24 is assessed as incidental and not treatment-related.

Haematological findings:

no effects observed

Description (incidence and severity):

No treatment-related changes among hematological parameters were observed.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

No treatment-related changes among clinical chemistry parameters were observed.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Uterus weights
The mean gravid uterus weight of the rabbits of test groups 1-3 (25, 75 or 250 mg/kg bw/d) was not influenced by the test substance. The differences between these groups and the control group showed no dose-dependency and were assessed to be without biological relevance.
For further details see supplementary table IA-012 in the attachement.

Absolute organ weights
When compared to control group 0 (set to 100%), the following absolute weights were increased:

Absolute weights
Test group 1 2 3
(mg/kg bw/d) (25) (75) (250)
Kidneys 105% 108%* 127%**
Liver 104% 107% 111%**
Spleen 135%** 119%* 121%*
*: p <= 0.05, **: p <= 0.01

All other mean absolute weight parameters did not show significant differences when compared to the control group 0.

Relative organ weights
When compared to control group 0 (set to 100%), the following mean relative weights were increased:

Relative weights
Test group 1 2 3
(mg/kg bw/d) (25) (75) (250)
Kidneys 102% 107%* 127%**
Liver 101% 105% 111%**
Spleen 132%** 117% 121%*
*: p <= 0.05, **: p <= 0.01

All other mean relative weight parameters did not show significant differences when com-pared to the control group 0.

Significantly increased absolute and relative kidney weights in test groups 2 and 3 as well as absolute and relative liver weights in test group 3 were regarded as treatment-related. Increased absolute and relative spleen weights were not dose-related and thus considered to be sporadic changes.

Increased absolute and relative kidney weights in test group 3 were above the range of historical control values.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

All findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.

Details on results:

Only pregnant does were used for the calculations of mean maternal food consumption, body weight and body weight change. Only pregnant does with scheduled sacrifice (GD 29) were used for the calculation of mean gravid uterine weights, mean organ weights, correct-ed (net) body weight gain and summary of reproduction data.
The following females were excluded from the above-mentioned calculations:
Test group 1 (25 mg/kg bw/d):
• female No. 35 - not pregnant
Test group 2 (75 mg/kg bw/d):
• female No. 59 - not pregnant
Test group 3 (250 mg/kg bw/d):
• female No. 84 - not pregnant
Thus, according to the requirements of the corresponding test guidelines, each test group including the controls contained a sufficient number of females with implantation sites at necropsy (approximately 20, but not fewer than 16 females with implantation sites).

Number of abortions:

no effects observed

Description (incidence and severity):

For further details see supplementary table IA-013 in the attachement.

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

For further details see supplementary table IA-013 in the attachement.

Total litter losses by resorption:

no effects observed

Description (incidence and severity):

For further details see supplementary table IA-013 in the attachement.

Early or late resorptions:

no effects observed

Description (incidence and severity):

For further details see supplementary table IA-014 in the attachement.

Dead fetuses:

effects observed, non-treatment-related

Description (incidence and severity):

For further details see supplementary table IA-013 in the attachement.

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

For further details see supplementary table IA-014 in the attachement.

Details on maternal toxic effects:

Female rabbits were placed into the study in four cohorts. Each dose group was represented in each cohort. The conception rate was 96% in the low-, mid- and high-dose groups (25, 75 and 250 mg/kg bw/d) and 100% in the control group. A sufficient number (approximately 20, but not fewer than 16 females with implantation sites) of pregnant females was available for the purpose of the study.

There were no test substance-related and/or biologically relevant differences between the different test groups in conception rate, in the mean numbers of corpora lutea and implantation sites or in the values calculated for the pre- and the post-implantation losses, the numbers of resorptions and viable fetuses. All differences observed are considered to reflect the normal range of fluctuations for animals of this strain and age.

One dead fetus, each, was found at cesarean section of control doe No. 7 (0 mg/kg bw/d) and high-dose doe No. 78 (250 mg/kg bw/d) which may occur spontaneously in this rabbit strain.

For further details see supplementary tables IA-013 and IA-014 in the attachement.

Dose descriptor:

NOAEL

Effect level:

75 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

body weight and weight gain

organ weights and organ / body weight ratios

Abnormalities:

no effects observed

Fetal body weight changes:

no effects observed

Description (incidence and severity):

The mean fetal weights of test groups 1, 2 and 3 were not influenced by the test substance and did not show any biologically relevant differences in comparison to the control group.

For further details see supplementary table ID-001 in the attachement.

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

For further details see supplementary table IA-015 in the attachement.

Changes in sex ratio:

no effects observed

Description (incidence and severity):

The sex distribution of the fetuses in test groups 1-3 (25, 75 and 250 mg/kg bw/d) was comparable to the control fetuses. Any observable differences were without biological relevance.

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

External malformations were detected in 4 low-dose fetuses of 2 different litters, in one mid-dose fetus as well as 3 high-dose fetuses of 3 different litters, as listed in the table below. Most of them were associated with skeletal malformations. However, these events added up to a statistically significantly increased total incidence of external malformations in test group 3. In absence of a dose-relationship and of a specific morphological pattern, none of these malformations were assessed as treatment-related.

Individual fetal external malformations

Test group Doe No.-Fetus No., Sex Finding
0 (0 mg/kg bw/d) none
1 (25 mg/kg bw/d) 29-03 F* spina bifida
29-05 M* thread-like tail
29-09 F* malrotated limb, spina bifida
47-04 F open eye
2 (75 mg/kg bw/d) 60-02 F umbilical hernia
3 (250 mg/kg bw/d) 77-05 F* mandible malformed
79-11 M* spina bifida
99-07 M* cleft palate

*fetus with additional skeletal malformations

Total external malformations

Test group 0 Test group 1 Test group 2 Test group 3
0 mg/kg bw/d 25 mg/kg bw/d 75 mg/kg bw/d 250 mg/kg bw/d
Litter N 25 24 24 24
Fetuses N 230 216 194 217
Fetal incidence N (%) 0.0 4 (1.9) 1 (0.5) 3 (1.4)
Litter incidence N (%) 0.0 2 (8.3) 1 (4.2) 3 (13)
Affected fetuses/litter Mean% 0.0 1.8 0.3 1.2*
* = p ≤ 0.05 (Wilcoxon-test [one-sided])


Fetal external variations
One external variation was recorded in one high-dose fetus (250 mg/kg bw/d), i.e. paw hyperflexion. This finding itself is reversible postnatally and not considered biologically relevant. It can be found in the historical control data.

Total external variations
Test group 0 Test group 1 Test group 2 Test group 3
0 mg/kg bw/d 25 mg/kg bw/d 75 mg/kg bw/d 250 mg/kg bw/d
Litter N 25 24 24 24
Fetuses N 230 216 194 217
Fetal incidence N (%) 0.0 0.0 0.0 1 (0.5)
Litter incidence N (%) 0.0 0.0 0.0 1 (4.2)
Affected fetuses/litter Mean% 0.0 0.0 0.0 0.5

Fetal external unclassified observations
Two unclassified external observations were recorded. Necrobiotic placentae were seen in five fetuses of high-dose litter No. 81 (250 mg/kg bw/d) and in one fetus of mid-dose litter No. 63 (75 mg/kg bw/d). This finding occurred exclusively in one litter, each, of the mid- and high-dose groups and, furthermore, can be found in the historical control data. Therefore it is not assessed as treatment-related. Furthermore, a skin tag was seen in one high-dose fetus (No. 86). This finding is not considered to be treatment-related.

Total external unclassified observations

Test group 0 Test group 1 Test group 2 Test group 3
0 mg/kg bw/d 25 mg/kg bw/d 75 mg/kg bw/d 250 mg/kg bw/d
Litter N 25 24 24 24
Fetuses N 230 216 194 217
Fetal incidence N (%) 0.0 0.0 1 (0.5) 6 (2.8)
Litter incidence N (%) 0.0 0.0 1 (4.2) 2 (8.3)
Affected fetuses/litter Mean% 0.0 0.0 0.7 3.0

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Fetal skeletal malformations
Skeletal malformations were recorded in single fetuses of all test groups including the control (0, 25, 75 or 250 mg/kg bw/d), as shown in the table below. Six fetuses had associated external malformations. Female low-dose fetus No. 29-03 had multiple skeletal malformations affecting the skull bones, vertbral column and ribs, while male mid-dose fetus No. 57-11 had multiple skeletal malformations concerning fore- and hindlimbs and the rib cage. All these findings were considered to be spontaneous in origin and not treatment-related.

No statistically significant differences between the groups were noted and no dose-response relationship was observed. The overall incidences were well within the historical control range of the test facility.

Individual fetal skeletal malformations

Test group Doe No.-Fetus No., Sex Finding
0 (0 mg/kg bw/d) 10-04 F malpositioned and bipartite sternebra
12-01 M additional vertebral arch and corresponding rib,
fused rib, intercostal rib
12-06 M severely malformed vertebral column and/or ribs
24-02 F absent lumbar vertebra
1 (25 mg/kg bw/d) 29-03 F* multiple skeletal malformations
29-05 M* severely malformed vertebral column and/or ribs
29-09 F* misshapen supraoccipital, splayed lumbar arch
38-03 M lumbar hemivertebra, absent rib
2 (75 mg/kg bw/d) 57-11 M multiple skeletal malformations
72-03 M misshapen thoracic vertebra, branched rib
3 (250 mg/kg bw/d) 77-05 F* fused mandible, short rib
77-15 M short rib
79-11 M* fused skull bone, splayed lumbar arch
82-01 M misshapen thoracic vertebra
91-11 F thoracic hemivertebra
96-11 F branched rib
99-07 M* small palatine bone
*fetus with additional external malformations

 
Tab. 4.3.4.1.2. Total skeletal malformations


Test group 0 Test group 1 Test group 2 Test group 3
0 mg/kg bw/d 25 mg/kg bw/d 75 mg/kg bw/d 250 mg/kg bw/d
Litter N 25 24 24 24
Fetuses N 230 216 194 217
Fetal incidence N (%) 4 (1.7) 4 (1.9) 2 (1.0) 7 (3.2)
Litter incidence N (%) 3 (12) 2 (8.3) 2 (8.3) 6 (25)
Affected fetuses/litter Mean% 2.0 1.7 0.9 2.9


Fetal skeletal variations
For all test groups, skeletal variations of different bone structures were observed, with or without effects on corresponding cartilages. The observed skeletal variations were related to several parts of fetal skeletons and appeared without a relation to dosing. The overall incidences of skeletal variations were comparable to the historical control data.

Total fetal skeletal variations
Test group 0 Test group 1 Test group 2 Test group 3
0 mg/kg bw/d 25 mg/kg bw/d 75 mg/kg bw/d 250 mg/kg bw/d
Litter N 25 24 24 24
Fetuses N 230 216 194 217
Fetal incidence N (%) 205 (89) 185 (86) 153 (79) 189 (87)
Litter incidence N (%) 25 (100) 24 (100) 24 (100) 24 (100)
Affected fetuses/litter Mean% 89.2 87.4 80.6 88.2


For a better overview, all skeletal variations with statistically significant differences between the control and the treated groups were compiled in the table belo. All incidences were expressed on a fetus per litter basis.

 
Occurrence of statistically significantly increased fetal skeletal variations (expressed as mean percentage of affected fetuses/litter)

Finding Test group 0 Test group 1 Test group 2 Test group 3 HCD
0 mg/kg bw/d 25 mg/kg bw/d 75 mg/kg bw/d 250 mg/kg bw/d Mean % (range)
Supernumerary
thoracic vertebra 11.6 25.3* 16.1 15.9 17.7 (7.8 - 25.6)
Fused sternebra;
unchanged cartilage 1.1 1.2 1.6 3.0* 1.3 (0.0 - 5.6)
Supernumerary rib;
cartilage present 47.0 63.4** 50.3 55.9 58.1 (50.0 - 70.9)
HCD = Historical control data
* = p ≤ 0.05 (Wilcoxon-test [one-sided]) ** = p ≤ 0.01 (Wilcoxon-test [one-sided])

As can be seen from the table above, the statistically significantly increased incidences of the listed skeletal variations were either not related to the dose and/or they were well inside the historical control range. Thus, they are not considered to be associated with treatment.


Fetal skeletal unclassified cartilage observations
Some isolated cartilage findings without impact on the respective bony structures, which were designated as unclassified cartilage observations, occurred in all test groups. The observed unclassified cartilage findings were related to the sternum and the ribs and did not show any relation to dosing. Therefore, they were assessed as not treat-ment-related.

Total unclassified cartilage observations
Test group 0 Test group 1 Test group 2 Test group 3
0 mg/kg bw/d 25 mg/kg bw/d 75 mg/kg bw/d 250 mg/kg bw/d
Litter N 25 24 24 24
Fetuses N 230 216 194 217
Fetal incidence N (%) 36 (16) 19 (8.8) 20 (10) 25 (12)
Litter incidence N (%) 16 (64) 9 (38) 11 (46) 11 (46)
Affected fetuses/litter Mean% 15.3 7.6 12.5 10.8

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Fetal soft tissue malformations
Soft tissue malformations occurred in all test groups including the control (0, 25, 75 or 250 mg/kg bw/d), as listed in the table below. Female mid-dose fetus No. 54-06 had multiple visceral malformations, i.e. fused and malpositioned kidneys, short ureter and absent gallbladder. The distribution of the findings about the test groups does not indicate an association to the treatment and no statistically significant differences between the groups were noted. The total incidence of soft tissue malformations in treated animals did not differ significantly from the control group and was comparable to the historical control data.

Individual fetal soft tissue malformations
Test group Doe No.-Fetus No., Sex Finding
0 (0 mg/kg bw/d) 3-02 F absent gallbladder
21-01 M absent subclavian
1 (25 mg/kg bw/d) 39-05 F, 39-06 M absent subclavian
2 (75 mg/kg bw/d) 54-06 F multiple visceral malformations
57-06 F small spleen
62-06 F absent subclavian
3 (250 mg/kg bw/d) 83-02 F absent kidney, absent ureter


Total soft tissue malformations
Test group 0 Test group 1 Test group 2 Test group 3
0 mg/kg bw/d 25 mg/kg bw/d 75 mg/kg bw/d 250 mg/kg bw/d
Litter N 25 24 24 24
Fetuses N 230 216 194 217
Litter
Fetal incidence N (%) 2 (0.9) 2 (0.9) 3 (1.5) 1 (0.5)
Litter incidence N (%) 2 (8.0) 1 (4.2) 3 (13) 1 (4.2)
Affected fetuses/litter Mean% 0.8 0.8 1.2 0.5
 
Fetal soft tissue variations
The examinations of the soft tissues revealed a broad variety of soft tissue variations, i.e. malpositioned carotid branch, narrowed aorta, dilated aorta, enlarged ventricular chamber of the heart and an absent lung lobe (Lobus inferior medialis) in individual fetuses of all test groups. The incidences of these variations were neither statistically significantly different from control nor dose-dependent and, therefore, not considered biologically relevant. Except ‘narrowed aorta’ which was seen in one control and one high-dose fetus, all findings can be found in the historical control data of the test facility at comparable incidences.

Total soft tissue variations
Test group 0 Test group 1 Test group 2 Test group 3
0 mg/kg bw/d 25 mg/kg bw/d 75 mg/kg bw/d 250 mg/kg bw/d
Litter N 25 24 24 24
Fetuses N 230 216 194 217
Fetal incidence N (%) 9 (3.9) 8 (3.7) 7 (3.6) 5 (2.3)
Litter incidence N (%) 4 (16) 6 (25) 4 (17) 4 (17)
Affected fetuses/litter Mean% 3.8 4.0 3.9 1.9

Fetal soft tissue unclassified observations
Four unclassified soft tissue observations were recorded. A blood coagulum around urinary bladder was seen in eleven control, three low-dose, one mid-dose and five high-dose fetus-es. This finding can be found in the historical control data at comparable incidences, therefore, it was neither assessed as treatment-related nor as adverse. Furthermore, a pale spleen and a pale liver were seen in two control fetuses of the same litter (No. 7), and hemorrhagic testis was recorded in two low-dose fetuses of different litters. These findings are not considered to be treatment-related.

Total soft tissue unclassified observations
Test group 0 Test group 1 Test group 2 Test group 3
0 mg/kg bw/d 25 mg/kg bw/d 75 mg/kg bw/d 250 mg/kg bw/d
Litter N 25 24 24 24
Fetuses N 230 216 194 217
Fetal incidence N (%) 13 (5.7) 5 (2.3) 1 (0.5) 5 (2.3)
Litter incidence N (%) 6 (24) 4 (17) 1 (4.2) 2 (8.3)
Affected fetuses/litter Mean% 4.7 2.8 0.7 3.6

Details on embryotoxic / teratogenic effects:

The mean placental weights in test groups 1, 2 and 3 were not influenced by the test substance and were similar to the control value.

There were noted external, soft tissue and skeletal malformations in all test groups including the control (0, 25, 75 or 250 mg/kg bw/d). The distribution of total malformations about the groups was not related to dose and at random.
Two fetuses of the control, four fetuses of the low-dose, three fetuses of the mid-dose and four fetuses of the high-dose group had more than one malformation or were multiple-malformed across the different examination areas. For a better overview, all those malformations were listed in the table below.

Fetuses with more than one malformation
Test group Doe No.-Fetus No., Sex Finding
0 (0 mg/kg bw/d) 12-01 M additional vertebral arch and corresponding rib,
fused rib, intercostal rib
12-06 M severely malformed vertebral column and/or ribs
1 (25 mg/kg bw/d) 29-03 F spina bifida, multiple skeletal malformations (small lumbar/sacral/caudal arches, fused skull bones, misshapen supraoccipital, fused thoracic arches, fused ribs)
29-05 M thread-like tail, severely malformed vertebral column and/or ribs
29-09 F malrotated limb, spina bifida, misshapen supraoccipital, splayed lumbar arch
38-03 M lumbar hemivertebra, absent rib
2 (75 mg/kg bw/d) 54-06 F multiple visceral malformations (fused and malpositioned kidneys, short ureter, absent gallbladder)
57-11 M multiple skeletal malformations (bent or broken fore /hindlimb bones, bent ribs)
72-03 M misshapen thoracic vertebra, branched rib
3 (250 mg/kg bw/d) 77-05 F mandible malformed, fused mandible, short rib
79-11 M spina bifida, fused skull bone, splayed lumbar arch
83-02 F absent kidney, absent ureter
99-07 M cleft palate, small palatine bone

Other malformations, such as umbilical hernia, open eye, absent subclavian, small spleen, absent gallbladder, thoracic hemivertebra, absent lumbar vertebra and malpositioned and bipartite sternebra were scattered observations in individual fetuses of all test groups including the control.
There was no statistically significant difference in the distribution of total malformations about the groups, and the incidences in the treated groups were inside the historical control range.
No morphologic or ontogenetic pattern is recognizable for the individual malformations or in the configuration of malformations in multiple-malformed fetuses, nor was there any cluster of any of these individual malformations seen in the other offspring of the test groups.
Overall, an association of all these findings to the treatment is not assumed.

Total fetal malformations
Test group 0 Test group 1 Test group 2 Test group 3
0 mg/kg bw/d 25 mg/kg bw/d 75 mg/kg bw/d 250 mg/kg bw/d
Litter N 25 24 24 24
Fetuses N 230 216 194 217
Fetal incidence N (%) 6 (2.6) 7 (3.2) 6 (3.1) 8 (3.7)
Litter incidence N (%) 5 (20) 4 (17) 5 (21) 7 (29)
Affected fetuses/litter Mean% 2.7 3.0 2.5 3.4

A spontaneous origin is assumed for the external variations, soft tissue variations and the broad range of skeletal variations which were observed in fetuses of all test groups including the controls.

If all different types of variations are summarized, none of the incidences showed a relation to dosing and can be found in the historical control data at a comparable frequency.

Total fetal variations
Test group 0 Test group 1 Test group 2 Test group 3
0 mg/kg bw/d 25 mg/kg bw/d 75 mg/kg bw/d 250 mg/kg bw/d
Litter N 25 24 24 24
Fetuses N 230 216 194 217
Fetal incidence N (%) 206 (90) 186 (86) 156 (80) 190 (88)
Litter incidence N (%) 25 (100) 24 (100) 24 (100) 24 (100)
Affected fetuses/litter Mean% 89.7 87.8 81.9 88.6


A spontaneous origin is assumed for the unclassified external, unclassified soft tissue and unclassified skeletal cartilage observations which were observed in several fetuses of test groups 0, 1, 2 and 3. The distribution and type of these findings do not suggest any relation to treatment.
Finally, fetal examinations revealed that there is no adverse effect of the compound on the respective morphological structures up to the highest dose tested (250 mg/kg bw/d).

For further details see supplementary tables ID-002-038 in the attachement.

Dose descriptor:

NOAEL

Effect level:

250 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Abnormalities:

effects observed, non-treatment-related

Developmental effects observed:

no

Lowest effective dose / conc.:

250 mg/kg bw/day (nominal)

For historical control data see supplementary section "3. Historical control data" in the attachement.

Conclusions:

Under the conditions of this prenatal developmental toxicity study, the oral administration of Isotridecanol to pregnant New Zealand White rabbits from implantation to one day prior to the expected day of parturition (GD 6-28) caused evidence of systemic maternal toxicity at the high-dose level of 250 mg/kg bw/d, such as decrease of food consumption and body weight/body weight gain during major parts of the treatment. In conclusion, the no observed adverse effect level (NOAEL) for maternal toxicity is 75 mg/kg bw/d.

As there was no evidence of toxicologically relevant adverse effects of the test substance on embryofetal development, the no observed adverse effect level (NOAEL) for prenatal de-velopmental toxicity is 250 mg/kg bw/d.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

750 mg/kg bw/day

Species:

rat

Additional information

Developmental toxicity of iso-Tridecan-1-ol (99.7%) was examined in a GLP-study (according to OECD 414), where 25 Wistar rats (per dose) were administered 60, 250 and 750 mg/kg bw iso-Tridecan-1-ol orally via gavage. The administration was daily from day 6 through day 19 post coitum (BASF, 2003). On gestation day 20, the dams were sacrificed and ovaries and uterine content as well as fetuses were examined. There were no substance-related or spontaneous mortalities in any of the groups. Some signs of maternal toxicity at 750 mg/kg body weight/day were detected. Clinically, transient salivation (in all rats), urine smeared fur (in 4 dams), and reduced food consumption at initiation of treatment was observed at the high dose level. Clinical pathology revealed increased alanine aminotransferase activities, decreased total protein and globulin concentrations, and increased triglyceride levels in the serum of the high dose females. These changes, which were accompanied by statistically significantly increased absolute and relative liver weights (about 14% or 18% respectively above control values), are indicative for a mild adverse effect on the liver. The only substance-induced finding on the mid dose dams (250 mg/kg body weight/day) consisted in transient salivation in 17 out of 25 rats, which, by is not assessed as an adverse or toxic effect, but rather due to the unpleasant taste of the substance. No substance-induced effects on the dams occurred at the low dose level (60 mg/kg body weight/day). The oral administration of iso-Tridecan-1-ol to the dams at all 3 dose levels (60, 250 and 750 mg/kg body weight/day) had no influence on the gestational parameters and evoked no signs of prenatal developmental toxicity and in particular no indications for teratogenicity at the 3 dose levels tested. Placental and fetal body weights were unaffected and the external, soft tissue and/or skeletal (including cartilage) examinations of the fetuses revealed no biologically relevant differences between the control and the substance-treated groups. Based on these results, the NOAEL for maternal toxicity is 250 mg/kg body weight/day, while it is 750 mg/kg body weight/day for prenatal developmental toxicity.

Additionally, Isotridecanol was administered to 25 pregnant New Zealand White rabbits (per dose) in a prenatal developmental toxicity study (according to OECD 414), daily by stomach tube from implantation to one day prior to the expected day of parturition (GD 6-28). Clinical observations and records of food consumption and body weight/body weight gain revealed no toxicologically relevant difference between the animals receiving 25 or 75 mg/kg bw/d Isotridecanol and the controls. The high dose of 250 mg/kg bw/d produced a decrease of food consumption as well as body weight/body weight gain during the first two of three treatment weeks. Although the animals partly compensated the lower food intake in the last treatment week, the high-dose does consumed overall 8% less food and had peak food consumption decreases of 31% compared to the concurrent control does during the treatment period (GD 6-28). Accordingly, lower food intake resulted in weight loss (-16.6 g vs. 28.4 g in the control) at the beginning of treatment, and the high-dose body weight remained below control during most of the treatment period. Only towards the end of treatment the high-dose mean body weights recovered and were comparable to the control again. If calculated for the treatment period (GD 6-28), the high-dose does gained about 8% less weight in comparison to the control does (without attaining statistical significance).  Concerning clinical pathology, no treatment-related, adverse effects were observed up to a dose of the test compound of 250 mg/kg bw/d. Concerning pathology, significantly increased absolute and relative kidney weights in test groups 2 and 3 as well as absolute and relative liver weights in test group 3 were regarded as treatment-related. 

There were no test substance-related and/or biologically relevant differences between thedifferent test groups in conception rate, in the mean numbers of corpora lutea and implantation sites or in the values calculated for the pre- and the post-implantation losses, the numbers of resorptions and viable fetuses. Similarly, no influence of the test substance on uterine weight, placental weight, fetal weight and sex distribution of the fetuses was noted at any dose. All differences observed are considered to reflect the normal range of fluctuations for animals of this strain and age. Fetal examinations revealed no toxicologically relevant adverse effects of the test substance on embryofetal development.

Under the conditions of this prenatal developmental toxicity study, the oral administration of Isotridecanol to pregnant New Zealand White rabbits from implantation to one day prior to the expected day of parturition (GD 6-28) caused evidence of systemic maternal toxicity at the high-dose level of 250 mg/kg bw/d, such as decrease of food consumption and body weight/body weight gain during major parts of the treatment. In conclusion, the no observed adverse effect level (NOAEL) for maternal toxicity is 75 mg/kg bw/d. As there was no evidence of toxicologically relevant adverse effects of the test substance on embryofetal development, the no observed adverse effect level (NOAEL) for prenatal developmental toxicity is 250 mg/kg bw/d.

Data for related long chain branched primary alcohols was also provided to support the read across justification as attached to IUCLID chapter 13.

Two compositions of branched isononyl alcohol, isodecanol, and propylheptanol were tested in a screening assay comparable to OECD 414, but using only 10 instead of 25 animals per dose group (BASF). All alcohols were tested at doses of 1, 5, and 10 mM/kg b.w. Isononanol was additionally tested at 7.5mM/kg b.w. The symptoms were comparable: Clinical signs consisted of unsteady gait, urine smeared fur, apathy, and, at higher doses, abdominal or lateral position. Body weight loss and reduced food consumption at the highest dose was observed during the first days of treatment (days 6 -10 p.c.). The first type of isononanol, consisting of several isomers with 2 methyl branches per molecule was more toxic than isononanol type 2, which consisted to about 50% of isomers with only one methyl or ethyl branch. Isononanol 1 killed all animals treated with 10mM/kg b.w. One dam died at 7.5mM/kg b.w. In comparison, 7 of the 10 Isononanol 2 treated rats of the high dose survived. Of the isodecanol treated rats, 4 did not survive in the high dose. Only one rat in the high dose treated with propylheptanol survived, but had no live fetuses. At lethal concentrations, an increased number of resorptions and lower fetal body weights were observed, which are considered secondary to severe maternal toxicity. The maternal NOAEL was 1mM/ kg b.w. for all alcohols described. No embryotoxicity with the exception of reduced body weights secondary to maternal body weight reduction or teratogenicity was observed with all alcohols at doses that were not lethal to the maternal animals.

Developmental toxicity of 2 -Propylhexan-1 -ol (99.8%) was also examined in a GLP-study according to OECD 414. 25 Wistar rats per dose were treated with 50, 200 and 600 mg/kg bw via gavage from day 6 through day 19 post coitum (BASF, 2004). At 600 mg/kg bw the following signs were recorded: salivation, reduced food consumption, reduced absolute and net body weight gain and increased water consumption. Organ weight determination revealed an increased absolute and relative liver weight. At the mid dose (200 mg/kg bw), the final body weight and body weight gain were still decreased, while the relative liver weight was slightly (5%) increased. The NOAEL for maternal toxicity was 50 mg/kg bw. Conception rate, mean number of corpora lutea, total implantations, resorptions and live fetuses, fetal sex ratio or values calculated for pre- and postimplantation losses were unaffected by the treatment. There was a statistically significant reduction of mean fetal body weight at the high dose level (600 mg/kg bw) of about 11% below the corresponding control value. Since this value was still in the range of historical control data, the finding is assessed to be secondary to the distinct maternal toxicity that has been observed in the appropriate dams. In the absence of teratogenic/embryotoxic effects, the NOAEL was 600 mg/kg bw for these parameters.

Nelson et al. (1988) examined the effects of isodecanol in an inhalation study similar to OECD guideline 414. 15 instead of 25 animals were used per group and a longer exposure period from gestation day 1 through 19 were employed. The only dose tested of 100mg/m³ had no effects on maternal toxicity, embryotoxicity, or teratogenicity and was thus the NOAEL.

Justification for classification or non-classification

The available data is conclusive. No classification according to 1272/2008/EC (CLP) criteria is required.