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Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
other: statistical reevaluation
Adequacy of study:
supporting study
Reliability:
other: statistical reevaluation
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Due to the fact that the fetuses are not statistically independent and to consider the variability between the litters, a one-sided Wilcoxon test (typically used for evaluation of fetal effects in current studies) was performed for the variation rate, the retardation rate and the variation + retardation rate. The mid dose group (650 mg/kg bw, g, test group 6) was compared to both water (test group 0) and vehicle control group (test group 1) and to the combined control group.

Data source

Referenceopen allclose all

Reference Type:
publication
Title:
Unnamed
Year:
1997
Report date:
1997
Reference Type:
other company data
Title:
Unnamed
Year:
1991
Report date:
1991
Reference Type:
study report
Title:
Unnamed
Year:
1991
Report date:
1991

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
other: comparative and thus limited study similar to EU Method B.31 (Prenatal Developmental Toxicity Study) (Cited as Directive 87/302/EEC, part B, p. 24)
Deviations:
yes
Remarks:
10 animals per group instead of 20
Qualifier:
according to guideline
Guideline:
other: comparative and thus limited study similar to OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
yes
Remarks:
10 animals per group instead of 20
GLP compliance:
yes

Test material

Constituent 1
Chemical structure
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
Details on test material:
- Analytical purity: >/= 99.5%
- Purity test: gas chromatogaphy

Test animals

Species:
rat
Strain:
Wistar
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Dr. K. Thomae GmbH, Biberach, Germany
- Age at study initiation: 68-85
- Weight at study initiation: 214-233
- Fasting period before study: no data
- Housing: singly, in stainless steel wire-mesh cages
- Diet: Kliba feed 343 ad libitum
- Water: ad libitum
- Acclimation period: at least 5 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 30-70
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Aqueous emulsions for gavage were freshly prepared every day under rapid stirring in doubly distileld water containing approx. 0.005% Cromophor EL

DIET PREPARATION
- Rate of preparation of diet (frequency): n.a
- Mixing appropriate amounts with (Type of food): n.a.
- Storage temperature of food: n.a.


VEHICLE
- Justification for use and choice of vehicle (if other than water): surfactant
- Concentration in vehicle:
- Amount of vehicle (if gavage): 0.005%
- Lot/batch no. (if required): no data
- Purity: no data
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Gas chromatography was used to verify test concentrations and stability during a 6-hr storage
Details on mating procedure:
- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1:1 to 1:4
- Length of cohabitation: until successful
- Verification of same strain and source of both sexes: yes
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
Duration of treatment / exposure:
gestation day 6 through 15
Frequency of treatment:
daily
Duration of test:
20 days
No. of animals per sex per dose:
10 females
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: equimolar doses of 6 different alcohols including 2-EH were trested at 0, 1, 5, and 10 mmol/kg bw.
- Rationale for animal assignment (if not random): random
- Other:

Examinations

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

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule:

BODY WEIGHT: Yes
- Time schedule for examinations: throughout the study

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): n.a., gavage study
- 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

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): n.a., gavage study
- Time schedule for examinations:

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


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: No data
Statistics:
For the fetal skeletal variations and retardation the Fisher’s exact test was performed and the fetus was taken as the statistical unit. Due to the fact that the fetuses are not statistically independent and to consider the variability between the litters, the litter should be taken as statistical unit. Therefore, a one-sided Wilcoxon test was performed for the variation rate, the retardation rate and the variation + retardation rate. The mid dose group (650 mg/kg bw) was compared to both water and vehicle control group and to the combined control group.
Indices:
No data published

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
1300 mg/kg bw/day: significant toxicity (discoloration of liver and lung; pronounced clinical symptoms (nasal discharge, salivation, CNS depression); reduced food consumption, body weight loss; 6 mortalities.
650 mg/kg bw/day: 2 damns with piloerection
130 mg/kg bw/day: no effect

Effect levels (maternal animals)

Dose descriptor:
NOAEL
Effect level:
650 mg/kg bw/day
Basis for effect level:
other: maternal toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
1300 mg/kg bw/day: increased early resorptions, high postimplantation loss; fetal body weights markedly reduced; increased incidences of skeletal malformations, variations, and retardations.
650 mg/kg bw/day: slightly decreased fetal weight; increased number of fetuses with skeletal variations and retardations
130 mg/kg bw/day: no effect

Effect levels (fetuses)

open allclose all
Dose descriptor:
NOAEL
Effect level:
650 mg/kg bw/day
Basis for effect level:
other: embryotoxicity
Dose descriptor:
NOAEL
Effect level:
650 mg/kg bw/day
Basis for effect level:
other: teratogenicity

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Any other information on results incl. tables

130 mg/kg dose group:
No adverse substance-related effects on dams or fetuses.

650 mg/kg dose group:
* maternal toxic effects
- 2 dams with piloerection
* embryo/fetotoxic effects
- slightly reduced mean fetal body weights ( 3.44 ± 0.28 g vs. 3.82 ± 0.23 g in control, i.e. 10%)

- increased frequency of fetuses with skeletal variations (vertebral column, sternum and ribs (27(7) / 65(9) fetuses (litters))

and retardations (incomplete or missing ossification of hyoid bone, vertebral bodies and sternebra(e) (51(9) / 65(9) fetuses (litters))


1300 mg/kg dose group:
* maternal toxic effects
- markedly reduced food consumption during the whole
treatment period (days 6-15 p.c.)
- distinctly reduced mean body weights (day 10 -20 p.c.)
body weight loss during days 6-10 p.c. and reduced body
body weight gains during days 10-15 p.c.; markedly reduced
corrected body weight gain
- 6 animals found dead on days 9, 10 and 13 p.c.
- severe clinical sypmtoms like abdominal or lateral
position, unsteady gait and apathy
- light brown-gray discoloration of the liver in the animals
with intercurrent death; lund edema and emphysema in a
few animals, and hemometra in 1 dam which showed vaginal
hemorrage before death
- distinctly reduced mean uterus weight
* embryo/fetotoxic effects
- increased number of resorptions and consequently markedly
increased postimplantation loss
- markedly reduced mean fetal body weights
- one fetus with acaudia and atresia ani; increased
incidence of fetuses with dilated renal pelvis and/or
hydroureter higher number of fetuses with skeletal
malformations, variations and retardations.

2 -EH: maternal and developmental toxicity (publication, table 5)
Dose levels (mg/kg bw/day)
0 (water)
0 (vehicle)
130
650
1300
No. pregnant dams
9
10
10
10
9
Fetuses and litters examined
124/9
146/10
130/10
127/9
28/2
Maternal deaths
1/10(a)
6/10
Pregnant at termination
9
10
10
10
9
Clinical signs
(+)
++
Body weight(g) day 0
231.5
230.8
229.6
225.5
235.6
Body weight (g) day 20
375.0
384.2
377.4
367.1
308.9**
Uterus weight (g)
77.7
82.2
72.2
75.9
32.9**
Corpora lutea /dam
16.1
16.0
15.4
15.7
15.3
Implantation sites/dam
15.0
15.7
13.6
14.8
14.8
Dams with viable fetuses
9
10
10
9
2
Postimplantation loss (%)
8.2
7.0
5.0
4.5
54.7**
Live fetuses/dam
13.8
14.6
13.0
14.1
14.0
Resorptions (total/early/dam)
1.2/1.1
1.1/1.0
0.6/0.5
0.7/0.2
7.8**/7.8**
Fetal weight (g)
3.80
3.82
3.80
3.44**
2.86**
No. (and %) of fetuses with malformations
1 (0.8)
2 (1.4)
3 (2.3)
7 (5.5) 
5** (17.9)
No. (and %) of litters with malformations (b)
1(11)
2(20)
3(30)
4(44)
2(100)
No. (and %) of fetuses with variations (b)
46 (37)
46 (32)
41(32)
49 (39)
20 (71)**
No. (and %) of litters with variations (b)
8(89)
10(100)
9(90)
8(89)
2(100)
No. (and %) of fetuses with retardations (b)
28(23)
38(26)
31(24)
51(40)
15(54)**
No. (and %) of litters with retardations (b)
8(89)
10(100)
8(80)
9(100)
2(100)


    

(a) due to gavage error       (b) percentage rounded

**p0.01

2-EH: incidence (%) and type of fetal findings; selected data (publication, tabe 6)


    
Dose levels (mg/kg bw/day)
0 (water)
0 (vehicle)
130
650
1300
No. of fetuses and litters examined
124/9
146/10
130/10
127/9
28/2
Dilated renal pelvis
23 (6)
28 (9)
18 (8)
21 (7)
10 (2)
Hydroureter
1
3 (2)
2 (2)
1
3
Total skeletal variations
23 (7)
17 (6)
23 (8)
27 (7)
10 (2)
Total skeletal retardations
28 (8)
38 (10)
31 (8)
51 (9)
15 (2)

Addendum:

In a subsequent assessment of the detailed study data the following observations were made:

650 mg/kg dose group:

* embryo/fetotoxic effects
- slightly reduced mean fetal body weights ( 3.44 ± 0.28 g vs. 3.82 ± 0.23 g in control, i.e. 10%)

BUT within the historical control range (3.9 ± 0.5 g; data from 302 litters)

- increased frequency of fetuses with skeletal variations (vertebral column, sternum and ribs (27(7) / 65(9) fetuses (litters))

and retardations (incomplete or missing ossification of hyoid bone, vertebral bodies and sternebra(e) (51(9) / 65(9) fetuses (litters))
      BUT skeletal variations and retardations were also observed at high incidences in vehicle control groups (e.g. 0.005% Cremophor EL)

- skeletal variation: sternum and ribs (17(6)/ 75(10) fetuses (litters));

- skeletal retardation: incomplete or missing ossification of vertebral bodies and sternebra(e) (38(10) / 75(10) fetuses (litters))

AND if the statistical evaluation is based on fetuses, the total incidence of skeletal variations (41.5%) slightly exceeds the historical control

value (37.4%); if the evaluation is based on the litter, the total incidence of skeletal variations (77.8%) is within the range of historical controls (88.9%)

OR if the statistical evaluation is based on the fetuses, the total incidence of skeletal retardation (78.5%) exceeds the historical control value (38.5%);

if the evaluation is based on the litter, the total incidence of skeletal retardation (100%; 9/9 litters) also exceeds the historical control value (83.9%)

AND Skeletal retardation in both control groups (dist. Water (43.1% of pups; 89% of litters) + Cremophor EL (50.7% of pups; 100% of litters)) also exceeds the historical control values.

Criticism of statistical analysis:

For statistical evaluation of fetal data, the Fisher exact test was used as stated in the study report. Due to the low litter size, the basis for statistical evaluation was the individual fetus, not the litter. Fetal responses within a dam are more similar than responses from different dams (known as “litter effect”). Thus, using the individual fetus data can lead to an inflation of Type I errors (implies “false positive results”).

Thus, statistical evaluation was performed again by using the Wilcoxon test (typically used for evaluation of fetal effects in current studies) on the basis of litter data. RESULT: using the Wilcoxon test, the statistical significance of the skeletal variations in the 650 mg/kg bw group could not be confirmed,

but the statistical significance of the skeletal retardation was still present (see also attached background material).

Current expert interpretation of effects in accordance with Regulation (EC) No 1272/200 section 3.7.2.3.3. ("If, in some reproductive toxicity studies in experimental animals the only effects recorded are considered to be of low or minimal toxicological significance, classification may not necessarily be the outcome. These effects include small changes in semen parameters or in the incidence of spontaneous defects in the foetus, small changes in the proportions of common foetal variants such as are observed in skeletal examinations, or in foetal weights, or small differences in postnatal developmental assessments."):

- In current developmental toxicity studies, skeletal variation and retardation are no longer considered as different effects. Thus, both effects were summarized and

statistical evaluation was performed again using Wilcoxon test.

Result: statistical significance was still confirmed.

- Skeletal retardation can be considered as non-adverse transient effects (Wilson et al.,1987; Collins et al., 1987; Marr et al., 1992; Holmbeck and Szabova, 2006; Carney and Kimmel, 2007)

Applicant's summary and conclusion

Conclusions:
In this oral prenatal developmental toxicity study in rats signs of developmental toxicity or teratogenicity, were only seen when maternal toxicity including mortality was present.
Executive summary:

The developmental toxicity of 2 -EH was investigated in a OECD TG 414 study conducted according to OECD TG 414 and under GLP, but using low rat numbers (10 instead of 20 pregnant females). This invalidates the study to some extend, but it provides weight of evidence for the developmental toxicity endpoint. Animals were treated on GD6 to 15 via gavage at concentrations of 130, 650, and 1300 mg/kg bw/d. Controls were either treated with water only or with vehicle (i.e. 0.005% Cremophor EL).

Maternal toxicity was most severe at the high dose level. In the mid and low dose no relevant maternal toxicity was noted. Therefore the NOAEL is 650 mg/kg/d for this endpoint under the conditions of this study.

Signs of embryo-/fetotoxicity were dose-dependently noted in dams showing signs of maternal toxicity at 1300 mg/kg/d. The observed effects (decrease mean fetal bw, increase of skeletal variations and retardation) in the mid-dose group were invalidated when compared to historical controls or current expert opinion in agreement with Regulation (EC) No 1272/2008 was applied:

- the decreased body weights were within the range of historical controls

- original statistical evaluation of the study was assessed to be invalid (reevaluation was performed by using Wilcoxon test)

- within this statistical reevaluation (based on fetus or litter size) the skeletal variations are rendered invalid (near or within historical controls)

- skeletal retardations are still significant

- however skeletal retardations of control goups also exceeded the historical control values

- and in agreement with current expert opinion and Regulation (EC) No 1272/2008 skeletal retardations can be considered as transient non-adverse effects.

Therefore the NOAEL is 650 mg/kg/d for this endpoint under the conditions of this study.

Teratogenicity was noted in fetuses from the high dose dams only. Therefore the NOAEL is 650 mg/kg/d under the conditions of this study for teratogenicity.

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