2-ethylhexyl nitrate

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Experiment using the rapidly formed hydrolysis product of the target chemical conducted according to a protocol comparable to guideline OECD TG 414; publication meets generally accepted standards and is reported in sufficient detail

Justification for type of information:

Source substance, 2-ethyl-1-hexanol is the rapidly formed hydrolysis product of the target substance, 2-EHN.

Data source

Reference

Reference Type:

publication

Title:

Developmental toxicology evaluation of 1-pentanol, 1-hexanol, and 2-ethyl-1-hexanol administered by inhalation to rats

Author:

BK Nelson, WS Brightwell, A Khan, EF Krieg, AM Hoberman

Year:

1989

Bibliographic source:

Journal of the American College of Toxicology, Vol 8, Number 2, 405-410

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

yes

Remarks:

15 female test animals (instead of 25)

GLP compliance:

not specified

Limit test:

yes

Test material

Test material information

Constituent 1

Reference substance name:

2-ethylhexan-1-ol

EC Number:

203-234-3

EC Name:

2-ethylhexan-1-ol

Cas Number:

104-76-7

Molecular formula:

C8H18O

IUPAC Name:

2-ethylhexan-1-ol

Test animals

Species:

rat

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratories, Portage, MI, U.S.A.
- Age at study initiation: No data
- Weight at study initiation: 200-300 g at beginning of pregnancy
- Fasting period before study: No
- Housing: singly, in stainless steel mesh wire cages during gestation and exposure
- Diet: Ad libitum (during exposure-free periods)
- Water: Ad libitum (during exposure-free periods)
- Acclimation period: 1-2 weeks

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

Administration / exposure

Route of administration:

inhalation: vapour

Type of inhalation exposure (if applicable):

whole body

Vehicle:

air

Remarks:

Constant flow of test item mixed with a defined volume of heated air

Details on exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Hinners-type 0.5 m³ exposure chamber
- Method of holding animals in test chamber: In cages
- Source and rate of air: Compressed ambient air
- Method of conditioning air: Heating
- Temperature and humidity in air chamber: 77±2 F° (i.e. 25°C), 50±15 %
- Air flow rate: 0.5 m³/min
- Air change rate: 60/h

TEST ATMOSPHERE
- Brief description of analytical method used: Hourly by infrared analyzer, twice a week by GC of charcoal tubes from the exposure chamber
- Samples taken from breathing zone: yes

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

- A Miran 1A infrared analyzer was constantly used for hourly measurement.
- Additionally the Exposure concentrations were
verified weekly by a secondary analysis. Charcoal tube samples were drawn 2 days/week and analyzed by gas chromatography, with partial verification by samples spiked with known concentrations

Details on mating procedure:

- Proof of pregnancy: Sperm in vaginal smear referred to as day 0

Duration of treatment / exposure:

Gestation days 1-19

Frequency of treatment:

7 h every day

Duration of test:

19 days (dams were sacrificed on day 20)

Doses / concentrations

Remarks:

Doses / Concentrations:
850 mg/m³ air
Basis:
analytical conc.

No. of animals per sex per dose:

15 (only females included)

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Probably in expectation of absence of effects, the experimental equipment was used to provide the highest achievable concentration while keeping the mixing chamber temperature below 80 °F (ca. 27 °C)>

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly

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

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No, calculated on a weekly basis

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

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes

Examinations included:
- Gravid uterus weight: Not reported
- 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 animals per litter
- Soft tissue examinations: Yes, half of the animals per litter
- Skeletal examinations: Yes, half of the animals per litter
- Head examinations: No

Statistics:

Multivariate analysis of variance (MANOVA) and analysis of variance (ANOVA)

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:

Maternal toxic effects:no effects. Remark: Overall feed consumption was approximatley 10-15% lower than in control animals.

Details on maternal toxic effects:
Although the weight gain appeared to be lower these difference did not reach statistical significance.

Effect levels (maternal animals)

1

Dose descriptor:

NOAEC

Remarks:

Discriminating level = sole test concentration, 7 h/day, gd 1-19

Effect level:

>= 850 mg/m³ air

Based on:

test mat.

Remarks:

2-Ethylhexan-1-ol

Basis for effect level:

other: developmental toxicity

2

Dose descriptor:

NOAEC

Remarks:

Discriminating level

Effect level:

>= 160 ppm

Based on:

test mat.

Remarks:

2-Ethylhexan-1-ol

Basis for effect level:

other: developmental toxicity

3

Dose descriptor:

NOAEC

Remarks:

Discriminating level

Effect level:

>= 1 148 mg/m³ air

Based on:

other: conversion to target chemical 2-Ethylhexyl Nitrate

Basis for effect level:

other: developmental toxicity

4

Dose descriptor:

NOAEC

Remarks:

Discriminating level

Effect level:

>= 6.5 other: mmol/m³

Based on:

other: molar concentration of source and target chemical

Basis for effect level:

other: developmental toxicity

5

Dose descriptor:

NOAEL

Remarks:

Discriminating dose

Effect level:

>= 193 other: mg/kg bw/day (inhalation-to-oral extrapolation)

Based on:

other: conversion to target chemical 2-Ethylhexyl Nitrate

Basis for effect level:

other: developmental toxicity

6

Dose descriptor:

NOAEL

Remarks:

Discriminating dose

Effect level:

>= 1.1 other: mmol/kg bw/day (inhalation-to-oral extrapolation)

Based on:

other: conversion to target chemical 2-Ethylhexyl Nitrate

Basis for effect level:

other: developmental toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
No indication of malformations induced after exposure of pregnant rats were found.

Effect levels (fetuses)

1

Dose descriptor:

NOAEC

Effect level:

850 mg/m³ air

Based on:

test mat.

Basis for effect level:

other: No toxicity to highest dose tested

2

Dose descriptor:

NOAEC

Effect level:

6.5 other: mmol/m3

Based on:

test mat.

Basis for effect level:

other: conversion to molar concentration to conver to 2-EHN concentration

3

Dose descriptor:

NOAEC

Effect level:

1 148 mg/m³ air

Based on:

act. ingr.

Basis for effect level:

other: conversion from molar concentration to 2-EHN concentration in mg/m3

Fetal abnormalities

Abnormalities:

not specified

Overall developmental toxicity

Developmental effects observed:

not specified

Any other information on results incl. tables

Table 1: Summary of data (Litter mean ± standard deviation)

Data	Test item 2-EH	Control
Mean maternal weight [g]
Day 0	283 ± 18	243 ± 25
Day 7	286 ± 17	262 ± 24
Day 14	310 ± 17	291 ± 26
Day 20	371 ± 20	354 ± 32
Overall gain	88	111
Mean feed consumption [g]
Week 1	97 ± 17	108 ± 14
Week 2	107 ± 12	124 ± 12
Week 3	113 ± 11	118 ± 10
Overall mean	106 ± 15 (a)	117 ± 13
Mean water intake [g]
Week 1	196 ± 32	204 ± 46
Week 2	203 ± 24	276 t 93
Week 3	248 ± 30	265 ± 123
Overall mean	216 ± 36	248 ± 96
Mean corpora lutea per litter	15 ± 2	14 ± 4
Mean resorptions per litter	0.3	0.4
Mean number females per litter	7 ± 2	8 ± 2
Mean number males per litter	7 ± 2	7 ± 2
Mean fetal weight [g]
Female	3.02 ± 0.20	3.19 ± 0.20
Male	3.18 ± 0.30	3.28 ± 0.27

(a) Significantly different from control

Applicant's summary and conclusion

Conclusions:

No maternal or foetal toxicity after inhalation exposure using the maximum vapour concentration of 850 mg/m³ air.

Executive summary:

The prenatal developmental toxicity of the source substance and test item 2-Ethylhexan-1-ol (CAS 104-76-7) via the inhalation route in the rat was measured in a GLP-compliant study using a “Prenatal Developmental Toxicity Study” compliant with OECD TG 414 (2001), with the sole deviation that the number of test animals was reduced (15 female test animals instead of 25). The validity criteria were met and the experiment can be considered relevant and adequate for the endpoint, however a reduced number of test animals were included. Nonetheless it is deemed conclusive and was rated „reliable with restrictions“, i.e. “Klimisch 2” according to the scale of Klimisch et al. (1997). In consideration of possible read-across from this study to metabolic precursors, additional uncertainties have to be taken into account. The rating of the study for use in such analogue or metabolite approach is lower compared to equally rated studies providing direct data from a target substance.

Fifteen sperm-positive female Sprague-Dawley rats were exposed to vapours of the test item through whole body exposure during gestation days 0 -19. Hinners type 0.5m³ exposure chambers to vapours of the test item at a concentration of 850 mg/m³ air was confirmed independently 2 methods; hourly by infrared analyzer and twice weekly by gas chromatography of charcoal tubes from the exposed chamber.

No maternal toxicity or developmental toxicity was noted in a rat inhalation study. The overall feed consumption was approximatley 10 -15% lower in treated animals compared to controls. Although the weight gain appeared to be lower in the treated animals, these differences did not reach statistical significance. The reproduction parameters were unchanged. No malformations were induced after exposure of the pregnant rats to the test substance. The incidences of resorptions, the number of fetuses per litter, the sex ratio, fetal weight, were not different to the control group and no external, skeletal or visceral malformations have been recorded.

In conclusion no substance-related adverse effects of the test item were observed at the highest vapour concentration achievable. Therefore the NOAEL (discriminating dose) was assigned to the highest test concentration. The NOAEL for maternal and developmental toxicity and teratogenicity in the rat is thus ≥ 850 mg/m³ air or ≥ 6.5 mmol/m³ as a basis for equimolar comparison in read-across approaches.

Reading-across to the target substance, 2-Ethylhexyl nitrate (2 -EHN, CAS 27247-96-7) delivers on the same molar concentration an air level of ≥1148 mg/m³ air. Taking the significantly lower vapour pressure of the target chemical (only 27 versus 93 Pa of 2 -EH at 20 °C) into account, an equimolar exposure exceeding saturated vapour concentrations of the target chemical can be concluded to be non-toxic during developmental exposure.

• Klimisch HJ, Andreae M, Tillmann U (1997). A Systematic Approach for Evaluating the Quality of Experimental Toxicological and Ecotoxicological Data. DOI 10.1006/rtph.1996.1076 PMID 9056496 Regul Toxicol Pharmacol 25:1-5.