Dipentaerythritol hexaesters of 3,5,5-trimethylhexanoic and n-heptanoic acids

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

There are no data available on toxicity to reproduction of Dipentaerythritol hexaesters of 3,5,5 -trimethylhexanoic and n-heptanoic acids. In order to fulfil the standard information requirements set out in Annex VIII, 8.7, in accordance with Annex XI, 1.5, of Regulation (EC) No 1907/2006, a read-across from structurally related substances was conducted.

Structural similarities and similarities in properties and/or activities of the source and target substance are the basis of read-across.

 

Toxicity to reproduction (fertility)

Dipentaerythritol hexaesters with 3,5,5,-trimethylhexanoic and n-heptanoic acids (EC 945-883-2, CAS 1379424-11-9 ) is considered to exhibit low toxicological activity based on the available studies with structural analogue substances, including a developmental toxicity study showing no adverse effects. In additon, as discussed in the toxicokinetic assessment, the substace is considered to have a very low potential for absorption.

Therefore, according to Regulation (EC) No 1907/2006 and with respect to animal welfare, further reproductive toxicity studies are not considered scientifically necessary.

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

the study does not need to be conducted because a pre-natal developmental toxicity study is available

Justification for type of information:

JUSTIFICATION FOR DATA WAIVING
[Specific explanation in addition to field 'Justification for data waiving']
Dipentaerythritol hexaesters with 3,5,5,-trimethylhexanoic and n-heptanoic acids is a substance of low toxicological activity, as other similar fatty esters of dipentaerythritol and pentaerythritol. Negative results for eye irritation, skin irritation and sensitization were obtained with the structural and chemical analogue substance Dipentaerythritol with fatty acids, C5 and C9iso (EC 444-000-2). The LD50 values found for acute toxicity by oral and dermal route (GLP studies OECD 423 and OECD 402 respectively) were both > 2000 mg/kg bw .
Furthermore, the subacute study (28 day, OECD 407, GLP) performed via the oral route did not reveal any adverse effects on reproductive organs and tissues including epididymis, ovaries, prostate, seminal vesicles, testes and uterus. No treatment-related effects on organ weight were noted. Males treated with 500 mg/kg bw/day showed a statistically significant reduction in absolute epididymis weight when compared with control but, in the absence of a convincing dose-response relationship, the intergroup difference was considered to be incidental and of no toxicological importance.
In addition, the prenatal developmental toxicity study (similar to OECD TG 414) with the analogue substance Trimethylolpropane caprylate caprate (CAS 11138-60-6, currently identified as EC 812-652-0 Fatty acids, C8-10-(even numbered), diesters and triesters with trimethylolpropane), via the dermal route did not show any treatment-related or adverse effects on intrauterine development and on reproductive organs and tissues (uterus and ovaries).
On the other side, as discussed in the toxicokinetic assessment, only a low potential for absorption is considered for the test substance while the limited amount of substance that may have become systemically available will hydrolyse and the breakdown cleavage products can be further metabolized.
In conclusion, Dipentaerythritol hexaesters with 3,5,5,-trimethylhexanoic and n-heptanoic acids (EC 945-883-2, CAS 1379424-11-9 ) is considered to exhibit low toxicological activity based on the available studies with structural analogue substances, including a developmental toxicity study showing no adverse effects.
Therefore, according to Regulation (EC) No 1907/2006 and with respect to animal welfare, further reproductive toxicity studies are not considered scientifically necessary.

Effect on fertility: via oral route

Endpoint conclusion:

no study available

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Effects on developmental toxicity

Description of key information

Developmental toxicity / teratogenicity

A Prenatal Developmental Toxicity Study was performed with structural analogue Trimethylolpropane caprylate caprate CAS 11138-60-6 (currently identified as EC 812-652-0 Fatty acids, C8-10-(even numbered), diesters and triesters with trimethylolpropane)via the dermal route equivalent or similar to OECD TG 414 (Azuka and Daston, 2004). The test substance was percutaneously applied to Sprague-Dawley rats for 6 h/day under occlusive conditions. 25 animals per sex per dose were treated with 200, 600 or 2000 mg/kg bw/day in corn oil on Days 6-15 of gestation. Control animals (25 per sex per dose) received the vehicle alone. The middle and the high dose levels caused some local irritation at the site of application, but no decreases in maternal weight gain or food consumption. There were no differences from control in any of the developmental parameters measured, including embryo/fetal viability, fetal weight, malformations, or variations. No adverse effects were observed in any maternal or repreoductive parameter or on external and skeletal development of fetuses.

Therefore, a NOAEL of 2000 mg/kg bw/day was derived for prenatal development and for systemic maternal toxicity. Due to the irritation effects on skin, the local maternal NOAEL was found to be 200 mg/kg bw/day.

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

Type of information:

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

Adequacy of study:

key study

Justification for type of information:

There is no data available on developmental toxicity of Dipentaerythritol hexaesters of 3,5,5-trimethylhexanoic and n-heptanoic acids (CAS 1379424 -11 -9). In order to fulfil the standard information requirements set out in Annex VIII, 8.7, in accordance with Annex XI, 1.5, of Regulation (EC) No 1907/2006, read-across from a structurally related substance was conducted. In accordance with Article 13 (1) of Regulation (EC) No 1907/2006, "information on intrinsic properties of substances may be generated by means other than tests, provided that the conditions set out in Annex XI are met.” In particular for human toxicity, information shall be generated whenever possible by means other than vertebrate animal tests, which includes the use of information from structurally related substances (grouping or read-across). Having regard to the general rules for grouping of substances and read-across approach laid down in Annex XI, Item 1.5, of Regulation (EC) No 1907/2006 whereby substances may be predicted as similar provided that their physicochemical, toxicological and ecotoxicological properties are likely to be similar or follow a regular pattern as a result of structural similarity.

The available information on toxicity for reproduction / developmental toxicity on substance Trimethylolpropane caprylate caprate (prevviously identified as CAS 11138-60-6) is used to predict the same behaviour for the target substance Dipentaerythritol hexaesters of 3,5,5-trimethylhexanoic and n-heptanoic acids (CAS 1379424-11-9).

The structural similarities between the source and the target substances are based on:

(1) common functional groups: The source (reference) and target substances are all characterised by ester bond(s) between a polyol and one or more carboxylic fatty acid chains. The polyol moiety of the source and target substances comprises structurally related molecules: pentaerythritol, di-pentaerythritol and trimethylolpropane, all of which share a neopentane backbone as underlying common molecular structure. The fatty acid moieties comprise saturated linear and/or branched chains of 5 to 10 C-atoms length.

(2) common precursors and the likelihood of common breakdown products via biological processes, which result in structurally similar chemicals. The source and target substance are all UVCB substances, produced by esterification of the corresponding polyol and fatty acid mixtures. Ester bond formation is in principle a reversible reaction (hydrolysis). A slow stepwise hydrolysis of the ester bonds by gastrointestinal enzymes is identified as the biological process, by which the breakdown of the source and target substances results in structurally similar chemicals: the respective polyol and fatty acid moieties as stated above.

(3) a constant pattern in the changing of the potency of the properties between source and target substances: For the source and target substances, the constant pattern is characterised by similarities in the potency of properties. The available data on the target and the source substance show similarities in physico-chemical properties. In particular, they are poorly soluble in water, have a high log Kow and high molecular weight.

The available data on toxicological properties indicate that the source and target substances have a similar toxicokinetic behaviour; especially they are assumed to be slowly hydrolyses (see toxicokinetics and attached data matrix).
In addition, the available data on genotoxicity show that Dipentaerythritol hexaesters of 3,5,5-trimethylhexanoic and n-heptanoic acids and the source substance Trimethylolpropane Caprylate Caprate are not genotoxic in the bacterial reverse mutation assay or clastogenic.

Available data on the teratogenic potential and influence on prenatal development of the structural surrogate source substance was considered for read-across and assessment was conducted based on an analogue approach (Azuka and Daston, 2004).
Decanoic acid, ester with 2-ethyl-2-(hydroxymethyl)-1,3-propanediol octanoate was tested in a prenatal developmental toxicity study equivalent to OECD guideline 414.
No effects on maternal body weight gain or food consumption were observed. In the mid- and high-dose group, local irritation at the site of application was apparent. Necropsy findings were limited to skin flaking and scabbing first identified in life and observations related to wearing the Elizabethan collar.
One dam of the mid-dose group (1/25) had a litter consisting of seven early resorptions. This result was pointed out as single non-dosage dependent event and to be within the historical ranges.
There were no significant differences from control in any of the developmental parameters measured. No differences were observed in the number of corpora lutea, implantation sites, pre- and post implantation loss, live and dead foetuses, resorptions and sex ratio among the groups, foetal weight, malformations, or variations. The observed effects in foetuses were dose-independent and regarded to be incidental and of no toxicological relevance.
Therefore, a NOAEL of 2000 mg/kg bw/day was derived for maternal toxicity and development toxicity. Furthermore, a local NOAEL of 200 mg/kg bw/day was derived, due to local irritation at the application site.

In summary, all available data on the source and target substances show that the constant pattern is characterised by a lack of potency of properties.

Reason / purpose for cross-reference:

read-across source

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Kingston, NY
- Age at study initiation: young adult
- Weight at study initiation: Mean of the maternal body weight: 226 g (Vehicle), 225 g (200 mg/kg bw/day), 227 g (600 mg/kg bw/day), 226 g (2000 mg/kg bw/day)
- Fasting period before study: No
- Housing: Virgin females were cohabitated with singly-housed male rats, one male per female rat for a maximum of 5 days and returned to individual housing in stainless steel wire-bottomed cages after mating.
- Diet: Certified Rodent Diet No. 5002 (PMI Feeds Inc. St.Louis, MO), ad libitum
- Water: water passaged through a reverse osmosis membrane with chlorine added as a bacteriostat, ad libitum
- Acclimation period: yes, period not mentioned

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 - 25
- Humidity (%): 30 - 70
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12 / 12

Route of administration:

dermal

Vehicle:

corn oil

Details on exposure:

TEST SITE
- Area of exposure: The back of the animals from the shoulders to the hip joints and extended ventrolaterally from the dorsal midline on each side (5x7 cm)
- % coverage: approx. 10% of the body surface
- Type of wrap: occlusive, gauze pad secured with Vetrap or Micropore tape
- Time intervals for shavings or clippings: during acclimatization period

REMOVAL OF TEST SUBSTANCE
- Washing: exposed area was wiped with a dermal wipe pad dampened with aqueous 1% solution of soap and then patted dry with a second clean pad
- Time after start of exposure: 6 h

TEST MATERIAL
- Amount(s) applied: 2 mL/kg
- Concentration: 200, 600, and 2000 mg/kg/day
- Constant volume or concentration used: yes

VEHICLE
- Amount(s) applied: 2 mL/kg
- Concentration: up to 100% (vehicle control)

USE OF RESTRAINERS FOR PREVENTING INGESTION: yes, Elizabethan collar

Analytical verification of doses or concentrations:

no

Details on mating procedure:

- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1/1
- Length of cohabitation: maximum 5 days
- Further matings after two unsuccessful attempts: Not applicable
- Verification of same strain and source of both sexes: No Data
- Proof of pregnancy: Both, vaginal plug and/or sperm in vaginal smear were referred to as Day 0 of pregnancy

Duration of treatment / exposure:

Treatment on Gestation Days (GD) 6 - 15

Frequency of treatment:

Daily

Duration of test:

Termination of the study by CO2 inhalation on GD 20.

No. of animals per sex per dose:

25

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dose dependent occurrence of skin irritation. Higher levels than 2000 mg/kg bw/day might be expected to produce marked irritation thereby compromising the interpretaion of developmental results.
- Rationale for animal assignment (if not random): Computer-generated randomization by weight (Barlett´s test for homogeneity) such that the groups were not statistically different (5% significance level) from each other.

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Animals were checked for mortality twice daily during the treatment period and daily thereafter.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Animals were checked for signs of reaction to treatment and/or symptoms of illness once daily before treatment, approx. 60 min after treatment during the dosing period. The dosing site was examined daily prior to substance application for signs of skin irritation according to Draize.

BODY WEIGHT: Yes
- Time schedule for examinations: Recorded on GD 0 and daily during the treatment period.

FOOD CONSUMPTION AND COMPOUND INTAKE : Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg b.w./day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation Day 20
- Organs examined: The uterus, uterine contents, position of the fetuses in the uterus and number of corpora lutea. Number and distribution of intrauterine implantations were classified as live or death fetuses, late intrauterine deaths (resorptions) and early intrauterine resorption sites. Live fetuses were sexed and further examined (see below).

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 (the heads of the animals used for soft tissue examinations)

Statistics:

Clinical observations and other proportion data were analyzed using the Variance Test for Homogeneity of the Binominal Distribution. Quantitative continuous data were analyzed using Barlett´s Test for Homogeneity of Variance and the Analysis of Variance when Barlett´s Test was not significant (p >0.05). If the Analysis of Variance was significant (p>0.05), Dunnett´s Test was used to identify the statistical significance of the individual groups. If the Analysis of Variance was not appropriate, the Kruskal-Wallis Test was used when >75% ties were present. In case of significance (p >0.05), Dunn´s Method of Multiple Comparisons was used for identification of statistical significance of the individual groups.

Historical control data:

No details. One dam having a litter consisting of seven early resorptions was pointed out as single non-dosage dependent event and to be within the ranges observed historically at the test facility.

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

effects observed, treatment-related

Description (incidence and severity):

The two highest dose levels caused some local irritation at the site of application, but no decreases in maternal weight gain or feed consumption (non-adverse)

Mortality:

mortality observed, non-treatment-related

Description (incidence):

Two animals in the control group and one animal in the high-dose group died within 6 h after first application; these were not considered to be treatment related and the animals were replaced (non-adverse)

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Necropsy findings were limited to skin flaking and scabbing first identified in life and observations related to wearing the Elizabethan collar (local alopecia, chromorhinorrhea, and neck lesions) (non-adverse)

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Total litter losses by resorption:

effects observed, non-treatment-related

Description (incidence and severity):

One dam of the mid-dose group (1/25) having a litter consisting of seven early resorptions was pointed out as single non-dosage dependent event and to be within the ranges observed historically at the test facility (non-adverse)

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Changes in pregnancy duration:

no effects observed

Changes in number of pregnant:

no effects observed

Other effects:

not examined

Details on maternal toxic effects:

Maternal toxic effects: yes (local irritation)

Details on maternal toxic effects:
The two highest dose levels caused some local irritation at the site of application, but no decreases in maternal weight gain or feed consumption. Two animals in the control group and one animal in the high-dose group died within 6 h after first application; these were not considered to be treatment related and the animals were replaced. One dam of the mid-dose group (1/25) having a litter consisting of seven early resorptions was pointed out as single non-dosage dependent event and to be within the ranges observed historically at the test facility.
Necropsy findings were limited to skin flaking and scabbing first identified in life and observations related to wearing the Elizabethan collar (local alopecia, chromorhinorrhea, and neck lesions).

Dose descriptor:

NOAEL

Effect level:

2 000 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: developmental toxicity

Dose descriptor:

NOAEL

Remarks:

local

Effect level:

200 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Dose descriptor:

NOAEL

Remarks:

systemic

Effect level:

2 000 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Fetal body weight changes:

no effects observed

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

no effects observed

Changes in postnatal survival:

not examined

External malformations:

no effects observed

Skeletal malformations:

no effects observed

Visceral malformations:

no effects observed

Other effects:

not examined

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects: no effects

Details on embryotoxic / teratogenic effects:
There were no significant differences from control in any of the developmental parameters measured, including embryo/fetal viability, fetal weight, malformations, or variations. The observed effects in fetuses were not dose-independent and regarded to be sporadic.

Remarks on result:

other: no effects determined

Abnormalities:

not specified

Developmental effects observed:

not specified

Table 1: Skin reaction observations

	0 mg/kg bw/d	200 mg/kg bw/d	600 mg/kg bw/d	2000 mg/kg bw/d
Maximum possible incidences^a	375/25	375/25	375/25	375/25
Erythema
Total	0/0	2/1	22/4	91/13^b
Grade 1	0/0	2/1	10/4	81/13^b
Grade 2	0/0	0/0	4/1	10/4^b
Flaking
Total	11/3	15/2	55/6	170/17 ^b
Grade 1	11/3	9/2	27/5	61/14 ^b
Grade 2	0/0	6/1	19/4	71/14^b
Grade 3	0/0	0/0	9/1	38/7 ^b
Edema
Total	0/0	0/0	23/4	83/11^b
Grade 1	0/0	0/0	18/4	59/11^b
Grade 2	0/0	0/0	5/1	24/6^b
Scab	0/0	0/0	6/2	19/4

a:       Maximum incidence : Days x rats from first treatment on GD 6 through sacrifice on GD 20 divided by the number of rats examined per group on GD 6-20

b:        Significantly different from vehicle control group value (p≤0.01)

 

Table 2: Maternal reproductive, litter, and fetal alteration observations: Caesarian-Section results on GD 20

	0 mg/kg bw/d	200 mg/kg bw/d	600 mg/kg bw/d	2000 mg/kg bw/d
Rats pregnant and sectioned on Day 20 of gestation (n)	25	23	22^b	24
Corpora lutea/dam	16.4	16.6	16.9	16.5
Implantation sites/litter	15.0	15.4	14.9	14.2
Litter size
Live fetuses/litter	14.6	14.6	14.0	13.3
Live fetuses (n)	364	335	308	320
Dead fetuses (n)	0	0	0	0
Resorptions	0.4	0.9	0.9	0.9
Early (n)	10	20	19	21
Late (n)	1	0	0	0
Dams with any resorptions n(%)	9 (36)	11 (48)	15 (68)	11 (46)
% resorbed/litter	2.9	5.4	5.8	5.0
% male/litter	51.3	50.8	48.1	47.7
Live fetal body weight (g/litter)	3.68	3.62	3.69	3.75
Male	3.77	3.68	3.82	3.85
Female	3.58	3.56	3.58	3.65
Fetuses evaluated (n)	364	335	308	320
Litters with any alterations observed n(%)	10 (40)	8 (35)	14 (64)	7 (25)
Fetuses with any alterations observed n(%)	13 (3.5)	10 (3.0)	20 (6.5)	9 (2.0)
% fetuses/litter with any alterations observed	3.5	2.9	6.8^c	2.7

b:       Excludes values for one dam, which had a litter consisting of seven early resorptions.

c:       Significantly different from vehicle control group value (p≤0.05)

Table 3: Fetal evaluations

	0 mg/kg bw/d	200 mg/kg bw/d	600 mg/kg bw/d	2000 mg/kg bw/d
Litters evaluated	25	23	22^b	24
Fetuses evaluated	364	335	308	320
Live	364	335	308	320
Fetal gross external alterations	364	335	308	320
Tail: kinked
Litter incidence, n (%)	0(0)	1 (4.3)	0(0)	0(0)
Fetal incidence, n (%)	0(0)	1(0.3)	0(0)	0(0)
Body: hematoma
Litter incidence, n (%)	1(4.0)	0(0)	0(0)	0(0)
Fetal incidence, n (%)	1 (0.3)	0(0)	0(0)	0(0)
Fetal soft tissue alterations, evaluations	174	162	149	155
Vessels: umbilical artery descended to the left of urinary bladder
Litter incidence, n (%)	2(8.0)	3(13.0)	2(9.1)	2(8.3)
Fetal incidence, n (%)	2(1.1)	3(1.8)	3(2.0)	2(1.3)
Vessels: apparent additional umbilical artery descended left of the bladder
Litter incidence, n (%)	0(0)	0(0)	1(4.5)	0(0)
Fetal incidence, n (%)	0(0)	0(0)	1(0.7)	0(0)
Fetal skeletal alterations, evaluations	190	173	159	165
Cervical vertebrae: cervical rib present at 7th cervical vertebrae
Litter incidence, n (%)	2(8.0)	1(4.3)	1(4.8)	0(0)
Fetal incidence, n (%)	2(1.0)	2(1.2)	1(1.2)	0(0)
Thoracic vertebrae: centrum, bifid
Litter incidence, n (%)	1(4.0)	1(4.3)	5(22.7)	0(0)
Fetal incidence, n (%)	1(0.5)	1(0.6)	5(3.1)^a	0(0)
Lumbar vertebrae: centrum, bifid
Litter incidence, n (%)	0(0)	1(4.3)	0(0)	0(0)
Fetal incidence, n (%)	0(0)	1(0.6)	0(0)	0(0)
Ribs: wavy
Litter incidence, n (%)	0(0)	0(0)	2(9.1)	1(4.2)
Fetal incidence, n (%)	0(0)	0(0)	2(1.2)	1(0.5
Sternal centra: 1st, not ossified
Litter incidence, n (%)	1(4.0)	0(0)	0(0)	2(8.3)
Fetal incidence, n (%)	1(0.5)	0(0)	0(0)	2(1.3)
Sternal centra: 1st, incompletely ossified
Litter incidence, n (%)	3(12.0)	3(13.0)	2(5.1)	1(4.2)
Fetal incidence, n (%)	4(2.1)	4(2.3)	2(1.2)	1(0.6)
Pelvis: pubis, incompletely ossified
Litter incidence, n (%)	3(12.0)	0(0)	4(18.2)	3(12.5)
Fetal incidence, n (%)	3(1.6)	0(0)	5(3.1)	3(1.8)
Pelvis: ischium, incompletely ossified
Litter incidence, n (%)	0(0)	0(0)	2(9.1)	0(0)
Fetal incidence, n (%)	0(0)	0(0)	2(1.2)	0(0)

a: Significantly different from vehicle control group (p≤0.01)

Conclusions:

In a developmental toxicity study, pregnant rats were dermally exposed to the test substance. No adverse effects were observed in any maternal or reproductive parameter or on external and skeletal development of fetuses. Thus, the developmental NOAEL (maternal/developmental) was determined to be 2000 mg/kg bw. The local NOAEL was considered to be 200 mg/kg bw/day due to local dermal irritation.

Reference 2

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

yes

Remarks:

few details on test substance given, no analysis of the test compound

GLP compliance:

not specified

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Kingston, NY
- Age at study initiation: young adult
- Weight at study initiation: Mean of the maternal body weight: 226 g (Vehicle), 225 g (200 mg/kg bw/day), 227 g (600 mg/kg bw/day), 226 g (2000 mg/kg bw/day)
- Fasting period before study: No
- Housing: Virgin females were cohabitated with singly-housed male rats, one male per female rat for a maximum of 5 days and returned to individual housing in stainless steel wire-bottomed cages after mating.
- Diet: Certified Rodent Diet No. 5002 (PMI Feeds Inc. St.Louis, MO), ad libitum
- Water: water passaged through a reverse osmosis membrane with chlorine added as a bacteriostat, ad libitum
- Acclimation period: yes, period not mentioned

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 - 25
- Humidity (%): 30 - 70
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12 / 12

Route of administration:

dermal

Vehicle:

corn oil

Details on exposure:

TEST SITE
- Area of exposure: The back of the animals from the shoulders to the hip joints and extended ventrolaterally from the dorsal midline on each side (5x7 cm)
- % coverage: approx. 10% of the body surface
- Type of wrap: occlusive, gauze pad secured with Vetrap or Micropore tape
- Time intervals for shavings or clippings: during acclimatization period

REMOVAL OF TEST SUBSTANCE
- Washing: exposed area was wiped with a dermal wipe pad dampened with aqueous 1% solution of soap and then patted dry with a second clean pad
- Time after start of exposure: 6 h

TEST MATERIAL
- Amount(s) applied: 2 mL/kg
- Concentration: 200, 600, and 2000 mg/kg/day
- Constant volume or concentration used: yes

VEHICLE
- Amount(s) applied: 2 mL/kg
- Concentration: up to 100% (vehicle control)

USE OF RESTRAINERS FOR PREVENTING INGESTION: yes, Elizabethan collar

Analytical verification of doses or concentrations:

no

Details on mating procedure:

- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1/1
- Length of cohabitation: maximum 5 days
- Further matings after two unsuccessful attempts: Not applicable
- Verification of same strain and source of both sexes: No Data
- Proof of pregnancy: Both, vaginal plug and/or sperm in vaginal smear were referred to as Day 0 of pregnancy

Duration of treatment / exposure:

Treatment on Gestation Days (GD) 6 - 15

Frequency of treatment:

Daily

Duration of test:

Termination of the study by CO2 inhalation on GD 20.

No. of animals per sex per dose:

25

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dose dependent occurrence of skin irritation. Higher levels than 2000 mg/kg bw/day might be expected to produce marked irritation thereby compromising the interpretaion of developmental results.
- Rationale for animal assignment (if not random): Computer-generated randomization by weight (Barlett´s test for homogeneity) such that the groups were not statistically different (5% significance level) from each other.

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Animals were checked for mortality twice daily during the treatment period and daily thereafter.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Animals were checked for signs of reaction to treatment and/or symptoms of illness once daily before treatment, approx. 60 min after treatment during the dosing period. The dosing site was examined daily prior to substance application for signs of skin irritation according to Draize.

BODY WEIGHT: Yes
- Time schedule for examinations: Recorded on GD 0 and daily during the treatment period.

FOOD CONSUMPTION AND COMPOUND INTAKE : Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg b.w./day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation Day 20
- Organs examined: The uterus, uterine contents, position of the fetuses in the uterus and number of corpora lutea. Number and distribution of intrauterine implantations were classified as live or death fetuses, late intrauterine deaths (resorptions) and early intrauterine resorption sites. Live fetuses were sexed and further examined (see below).

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 (the heads of the animals used for soft tissue examinations)

Statistics:

Clinical observations and other proportion data were analyzed using the Variance Test for Homogeneity of the Binominal Distribution. Quantitative continuous data were analyzed using Barlett´s Test for Homogeneity of Variance and the Analysis of Variance when Barlett´s Test was not significant (p >0.05). If the Analysis of Variance was significant (p>0.05), Dunnett´s Test was used to identify the statistical significance of the individual groups. If the Analysis of Variance was not appropriate, the Kruskal-Wallis Test was used when >75% ties were present. In case of significance (p >0.05), Dunn´s Method of Multiple Comparisons was used for identification of statistical significance of the individual groups.

Historical control data:

No details. One dam having a litter consisting of seven early resorptions was pointed out as single non-dosage dependent event and to be within the ranges observed historically at the test facility.

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

effects observed, treatment-related

Description (incidence and severity):

The two highest dose levels caused some local irritation at the site of application, but no decreases in maternal weight gain or feed consumption (non-adverse)

Mortality:

mortality observed, non-treatment-related

Description (incidence):

Two animals in the control group and one animal in the high-dose group died within 6 h after first application; these were not considered to be treatment related and the animals were replaced (non-adverse)

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Necropsy findings were limited to skin flaking and scabbing first identified in life and observations related to wearing the Elizabethan collar (local alopecia, chromorhinorrhea, and neck lesions) (non-adverse)

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Total litter losses by resorption:

effects observed, non-treatment-related

Description (incidence and severity):

One dam of the mid-dose group (1/25) having a litter consisting of seven early resorptions was pointed out as single non-dosage dependent event and to be within the ranges observed historically at the test facility (non-adverse)

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Changes in pregnancy duration:

no effects observed

Changes in number of pregnant:

no effects observed

Other effects:

not examined

Details on maternal toxic effects:

Maternal toxic effects: yes (local irritation)

Details on maternal toxic effects:
The two highest dose levels caused some local irritation at the site of application, but no decreases in maternal weight gain or feed consumption. Two animals in the control group and one animal in the high-dose group died within 6 h after first application; these were not considered to be treatment related and the animals were replaced. One dam of the mid-dose group (1/25) having a litter consisting of seven early resorptions was pointed out as single non-dosage dependent event and to be within the ranges observed historically at the test facility.
Necropsy findings were limited to skin flaking and scabbing first identified in life and observations related to wearing the Elizabethan collar (local alopecia, chromorhinorrhea, and neck lesions).

Dose descriptor:

NOAEL

Effect level:

2 000 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: developmental toxicity

Dose descriptor:

NOAEL

Remarks:

local

Effect level:

200 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Dose descriptor:

NOAEL

Remarks:

systemic

Effect level:

2 000 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Fetal body weight changes:

no effects observed

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

no effects observed

Changes in postnatal survival:

not examined

External malformations:

no effects observed

Skeletal malformations:

no effects observed

Visceral malformations:

no effects observed

Description (incidence and severity):

An increased number of fetuses with levocardia was observed after in utero exposure to TMP ester of C8/C10 fatty acids (ESIS: Decanoic acid, ester with 2-ethyl-2-(hydroxymethyl)-1,3-propanediol octanoate) (CAS 11138-60-6) via the dermal route. However, as no malformations in the heart were detected and levocardia was also observed in control fetuses of the respective test facility and in a further study (Smith et al. 1988), the toxicological relevance of this effect remains questionable. Thus, incidental occurrence of levocardia cannot be excluded.

Other effects:

not examined

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects: no effects
There were no significant differences from control in any of the developmental parameters measured, including embryo/fetal viability, fetal weight, malformations, or variations. The observed effects in fetuses were not dose-independent and regarded to be sporadic.

Remarks on result:

other: no effects determined

Abnormalities:

not specified

Developmental effects observed:

not specified

Table 1: Skin reaction observations

	0 mg/kg bw/d	200 mg/kg bw/d	600 mg/kg bw/d	2000 mg/kg bw/d
Maximum possible incidences^a	375/25	375/25	375/25	375/25
Erythema
Total	0/0	2/1	22/4	91/13^b
Grade 1	0/0	2/1	10/4	81/13^b
Grade 2	0/0	0/0	4/1	10/4^b
Flaking
Total	11/3	15/2	55/6	170/17 ^b
Grade 1	11/3	9/2	27/5	61/14 ^b
Grade 2	0/0	6/1	19/4	71/14^b
Grade 3	0/0	0/0	9/1	38/7 ^b
Edema
Total	0/0	0/0	23/4	83/11^b
Grade 1	0/0	0/0	18/4	59/11^b
Grade 2	0/0	0/0	5/1	24/6^b
Scab	0/0	0/0	6/2	19/4

a:       Maximum incidence : Days x rats from first treatment on GD 6 through sacrifice on GD 20 divided by the number of rats examined per group on GD 6-20

b:        Significantly different from vehicle control group value (p≤0.01)

 

Table 2: Maternal reproductive, litter, and fetal alteration observations: Caesarian-Section results on GD 20

	0 mg/kg bw/d	200 mg/kg bw/d	600 mg/kg bw/d	2000 mg/kg bw/d
Rats pregnant and sectioned on Day 20 of gestation (n)	25	23	22^b	24
Corpora lutea/dam	16.4	16.6	16.9	16.5
Implantation sites/litter	15.0	15.4	14.9	14.2
Litter size
Live fetuses/litter	14.6	14.6	14.0	13.3
Live fetuses (n)	364	335	308	320
Dead fetuses (n)	0	0	0	0
Resorptions	0.4	0.9	0.9	0.9
Early (n)	10	20	19	21
Late (n)	1	0	0	0
Dams with any resorptions n(%)	9 (36)	11 (48)	15 (68)	11 (46)
% resorbed/litter	2.9	5.4	5.8	5.0
% male/litter	51.3	50.8	48.1	47.7
Live fetal body weight (g/litter)	3.68	3.62	3.69	3.75
Male	3.77	3.68	3.82	3.85
Female	3.58	3.56	3.58	3.65
Fetuses evaluated (n)	364	335	308	320
Litters with any alterations observed n(%)	10 (40)	8 (35)	14 (64)	7 (25)
Fetuses with any alterations observed n(%)	13 (3.5)	10 (3.0)	20 (6.5)	9 (2.0)
% fetuses/litter with any alterations observed	3.5	2.9	6.8^c	2.7

b:       Excludes values for one dam, which had a litter consisting of seven early resorptions.

c:       Significantly different from vehicle control group value (p≤0.05)

Table 3: Fetal evaluations

	0 mg/kg bw/d	200 mg/kg bw/d	600 mg/kg bw/d	2000 mg/kg bw/d
Litters evaluated	25	23	22^b	24
Fetuses evaluated	364	335	308	320
Live	364	335	308	320
Fetal gross external alterations	364	335	308	320
Tail: kinked
Litter incidence, n (%)	0(0)	1 (4.3)	0(0)	0(0)
Fetal incidence, n (%)	0(0)	1(0.3)	0(0)	0(0)
Body: hematoma
Litter incidence, n (%)	1(4.0)	0(0)	0(0)	0(0)
Fetal incidence, n (%)	1 (0.3)	0(0)	0(0)	0(0)
Fetal soft tissue alterations, evaluations	174	162	149	155
Vessels: umbilical artery descended to the left of urinary bladder
Litter incidence, n (%)	2(8.0)	3(13.0)	2(9.1)	2(8.3)
Fetal incidence, n (%)	2(1.1)	3(1.8)	3(2.0)	2(1.3)
Vessels: apparent additional umbilical artery descended left of the bladder
Litter incidence, n (%)	0(0)	0(0)	1(4.5)	0(0)
Fetal incidence, n (%)	0(0)	0(0)	1(0.7)	0(0)
Fetal skeletal alterations, evaluations	190	173	159	165
Cervical vertebrae: cervical rib present at 7th cervical vertebrae
Litter incidence, n (%)	2(8.0)	1(4.3)	1(4.8)	0(0)
Fetal incidence, n (%)	2(1.0)	2(1.2)	1(1.2)	0(0)
Thoracic vertebrae: centrum, bifid
Litter incidence, n (%)	1(4.0)	1(4.3)	5(22.7)	0(0)
Fetal incidence, n (%)	1(0.5)	1(0.6)	5(3.1)^a	0(0)
Lumbar vertebrae: centrum, bifid
Litter incidence, n (%)	0(0)	1(4.3)	0(0)	0(0)
Fetal incidence, n (%)	0(0)	1(0.6)	0(0)	0(0)
Ribs: wavy
Litter incidence, n (%)	0(0)	0(0)	2(9.1)	1(4.2)
Fetal incidence, n (%)	0(0)	0(0)	2(1.2)	1(0.5
Sternal centra: 1st, not ossified
Litter incidence, n (%)	1(4.0)	0(0)	0(0)	2(8.3)
Fetal incidence, n (%)	1(0.5)	0(0)	0(0)	2(1.3)
Sternal centra: 1st, incompletely ossified
Litter incidence, n (%)	3(12.0)	3(13.0)	2(5.1)	1(4.2)
Fetal incidence, n (%)	4(2.1)	4(2.3)	2(1.2)	1(0.6)
Pelvis: pubis, incompletely ossified
Litter incidence, n (%)	3(12.0)	0(0)	4(18.2)	3(12.5)
Fetal incidence, n (%)	3(1.6)	0(0)	5(3.1)	3(1.8)
Pelvis: ischium, incompletely ossified
Litter incidence, n (%)	0(0)	0(0)	2(9.1)	0(0)
Fetal incidence, n (%)	0(0)	0(0)	2(1.2)	0(0)

a: Significantly different from vehicle control group (p≤0.01)

Conclusions:

In a developmental toxicity study, pregnant rats were dermally exposed to the test substance. No adverse effects were observed in any maternal or reproductive parameter or on external and skeletal development of fetuses. Thus, the developmental NOAEL (maternal/developmental) was determined to be 2000 mg/kg bw. The local NOAEL was considered to be 200 mg/kg bw/day due to local dermal irritation.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

2 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

The available information comprises an adequate and reliable study (Klimisch score 2) from reference substance with similar structure and intrinsic properties. Read-across is justified based on common functional group(s), common precursors/breakdown products, similarities in PC/ECO/TOX properties (refer to endpoint discussion for further details).

Justification for classification or non-classification

Based on the analogue read-across approach, the available data on developmental toxicity do not meet the classification criteria according to Regulation (EC) 1272/2008, and are therefore conclusive but not sufficient for classification.