Hexa-2,4-dienoic acid

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study (GLP)

Data source

Reference

Reference 1

Reference Type:

study report

Title:

Unnamed

Year:

2004

Report date:

2004

Reference 2

Reference Type:

study report

Title:

Unnamed

Year:

2004

Report date:

2004

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

adopted 2001-01-22

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.31 (Prenatal Developmental Toxicity Study)

Version / remarks:

1988

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

signed 2003-12-08

Test material

Test material information

Constituent 1

Reference substance name:

Hexa-2,4-dienoic acid

EC Number:

203-768-7

EC Name:

Hexa-2,4-dienoic acid

Cas Number:

110-44-1

Molecular formula:

C6H8O2

IUPAC Name:

hexa-2,4-dienoic acid

Test material form:

solid

Details on test material:

- Name of test material: Sorbic acid
- Physical state: White, crystalline powder
- Lot/batch No.: 0000009738
- Expiration date of the lot/batch (retest date): 2006-03-27

Specific details on test material used for the study:

Batch No. 0000009738
Purity: 99.9%
Appearance: white, crystalline power

Test animals

Species:

rabbit

Strain:

Himalayan

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: LPT, Löhndorf/Wankendorf
- Age at study initiation: 4 to 5 month
- Weight at study initiation: 2.20 to 2.89 kg.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: 0.5 % aqueous hydroxypropyl-methylcellulose gel

Details on exposure:

TOTAL VOLUME APPLIED: 10 mL/kg bw/day

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Recovery was invariably better than 80 % of nominal, indicating that doses were close to nominal and homogeneousyl distributed in the matrix.

Details on mating procedure:

Mating period: Caging of one male and one female. The day of copulation was considered as day 0 of pregnancy.

Duration of treatment / exposure:

Duration of exposure: Day 6-29 of gestation

Frequency of treatment:

Daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day

Remarks:

Control

Doses / concentrations 2

Dose / conc.:

300 mg/kg bw/day

Doses / concentrations 3

Dose / conc.:

1 000 mg/kg bw/day

Doses / concentrations 4

Dose / conc.:

3 000 mg/kg bw/day

No. of animals per sex per dose:

Number of animals per group: 20 females (17 for the high dose group)
Control animals: 20 females

Control animals:

yes, concurrent vehicle

Details on study design:

Post-exposure period: None

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: daily

FOOD CONSUMPTION: Yes
- Time schedule: daily

WATER CONSUMPTION: Yes
- Time schedule for examinations: daily

OTHER: Macroscopic examination of the internal organs was performed upon necropsy of the dams.

Ovaries and uterine content:

Examination of uterine content: Gravid uterine weight, no. of corpora  lutea, pre- and post-implantation loss, early and late resorptions

Fetal examinations:

Examination of foetuses: 
- General: Litter size, no. of dead and alive foetuses, no. and weight of placentae, viability of foetuses, foetal  weight, sex ratio, external abnormalities.
- Skeleton: Yes
- Soft tissue: Yes

Statistics:

For all numerical values, homogeneity of variances was tested using the Bartlett chi-square test. When the variances were homogeneous, the Dunnett test (p ~ 0.01) and for heterogeneity of variances, the Student's t-test was carried out. For comparisons, FISHER's exact test(n < 100)
or chi2-test with Yates' correction for continuity (n ~ 100) (p :'.': 0.05 and p ~ 0.01}

Historical control data:

Reference to the existing historical control: The relationship between 'Variations and Retardations' and 'Malformations' should be seen under the aspect of cumulative historic data.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

At 300, 1000 and 3000 mg/kg b.w./day, slightly reduced motility was noted in all dams, starting within 20 to 60 minutes after administration. In addition, an increased respiratory rate was noted at 1000 and 3000 mg/kg b. w ./day. These symptoms lasted - in a dose-related way - for up to 60 minutes in the low-dosed dams, and for up to 2 hours in the intermediate-dosed and in the high-dosed dams.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

No test item-related mortality occurred for the dams treated with 300 or 1000 mg/kg b. w ./
day.
At 3000 mg/kg b.w./day, 8 females died prematurely between gestation day 8 and 28,
predominantly unobserved during the night. In addition, 8 dams were sacrificed after abortion
between gestation day 17 and 28. Necropsy of the prematurely deceased dams revealed
changes of the spleen (coarse-grained rough surface, reduced in size) and gastric lesions
(hemorrhagic foci) in all animals.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

At 1000 mg/kg b. w ./day, a marginal but statistically significant reduction (at p :s: 0.05) of mean body weight gain was noted for the period of gestation days 6 to 9.
At 3000 mg/kg b.w./day, the mean maternal body weight was slightly reduced during the
treatment period, statistically significant reductions (at p :s: 0.05) were noted on gestation
days 16, 21 and 25 to 29 (up to 6% below the control value). Body weight gain was statistically significantly (p ~ 0.01) reduced during the periods of gestation days 6 to 9, 9 to 12, and 12 to 15.

Food efficiency:

effects observed, treatment-related

Description (incidence and severity):

A dose-related reduced food consumption was observed starting at 300 mg/kg b. w ./day.
At 3000 mg/kg b.w./day, absolute and relative food intake were markedly reduced during the
treatment period up to 63 % compared to the control value.

Ophthalmological findings:

not specified

Haematological findings:

not specified

Clinical biochemistry findings:

not specified

Endocrine findings:

not examined

Urinalysis findings:

not specified

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

The gravid uterus weight, the carcass weight and the net weight change from day 6 onwards {carcass weight minus day 6 body weight) of the animals treated with 300 or 1000 mg/kg b. w ./day were not influenced by the exposure to the test item.
At 3000 mg/kg b.w./day, a statistically significant reduction was noted for the gravid uterus
weight (minus 52% below the control value).

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No test item-related pathological findings were noted at macroscopic inspection in the dams treated with 300 mg/kg b.w./day. Necropsy revealed a black focus at one placenta (diameter approx. 8 mm) of dam no. 32 (fetus no. 3). This finding is regarded as spontaneous.
At 1000 mg/kg b.w./day, necropsy revealed a (coarse-grained) rough surface of
the spleen in all intermediate-dosed dams. This finding is regarded as test itemrelated.
In addition, a dark-grey discoloured, partly indurated placenta was noted in dam
no. 53 (fetus no. 4), this finding was judged as spontaneous.
Laparotomy revealed that two dams (nos. 49 and 50) had obviously endured an
unobserved abortion, these females were excluded from statistics. This incidence
is considered to be within the normal range, one abortion was also noted in the
control group.
At 3000 mg/kg b.w./day, necropsy revealed macroscopic test item-related
changes of the spleen (coarse-grained rough surface, reduced in size) in nearly all
high-dosed dams including prematurely deceased animals and animals which
endured an abortion. No test item-related lesions were noted at the gastric
mucosa or small and large intestines of the surviving animals.
A pale liver and a haemorrhagic area at the outer surface of the appendix were
noted in one dam with abortion (no. 79).
Necropsy of the prematurely deceased dams revealed gastric lesions (stomach:
haemorrhagic/dark-red foci, ulcers or reddenings) in all animals and intestinal
changes (intestines: dark-red contents) in one of these dams. Further findings
such as emaciation, dark-red lungs and a discoloured (light-red) heart were noted
in one or two deceased animals.
In total, eleven dams endured an abortion, these females were excluded from
statistics. In four dams (nos. 74, 78, 87 and 95) the abortion had not been
observed during gestation, but was detected at laparotomy.

Neuropathological findings:

not specified

Histopathological findings: non-neoplastic:

not specified

Histopathological findings: neoplastic:

not specified

Maternal developmental toxicity

Number of abortions:

effects observed, treatment-related

Description (incidence and severity):

The abortions observed in this study are regarded to be test-item related at 3000 mg/kg b.w./day.

Pre- and post-implantation loss:

effects observed, treatment-related

Description (incidence and severity):

No effects in the number of pre-implantation in all treated groups. in 300 and 1000 mg/kg bw/ day. However, Regarding post-implantation loss, at 3000 mg/kg bw/day it was increased 29.2% vs. control 4.5%.

Total litter losses by resorption:

not specified

Early or late resorptions:

effects observed, treatment-related

Description (incidence and severity):

There were no test item-related biological differences between the control group and the animals treated either with 300 or 1000 mg/kg b. w ./day in the number of resorptions and live fetuses or the values calculated for the pre- and post-implantation losses.

The number of early and total resorptions was significantly increased at p ~ 0.01 (29 total
resorptions in the highest dose (3000 mg/kg bw/day), 6 resorptions in the control). The number of
fetuses was accordingly statistically decreased.

Dead fetuses:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item-related biological differences between the control group and the animals treated either with 300 or 1000 mg/kg b. w./day in the number of live fetuses. Also, at 3000 mg/kg bw/day the number of fetuses was accordingly statistically decreased.

Changes in pregnancy duration:

effects observed, non-treatment-related

Changes in number of pregnant:

effects observed, treatment-related

Description (incidence and severity):

A number of aborts were seen at 3000 mg/kg bw/day (21%).

Details on maternal toxic effects:

Maternal toxic effects:yes

Details on maternal toxic effects:
Shortly following administration of 300, 1000 or 3000 mg/kg bw/day slightly reduced motility was observed for up to 2 hours. This finding  was accompanied by an increased respiratory rate in mid and high dose  dams. No dams in the low or mid dose group died prematurely. Administration of 3000 mg/kg bw/day caused death in 8 females and a further 11 females aborted. No effect on body weight was observed in the low  dose group. At 1000 mg/kg bw/day, marginal statistically significant  reduction of mean body weight gain was observed for gestation days 6 to 9. At 3000 mg/kg bw/day, maternal body weight was slightly reduced during  the treatment period, reaching statistical significance on days 16, 21, and 25 to 29 of gestation. The mean maternal body weight change was  statistically significant decreased on gestation days 6 to 9, 9 to 12 and  12 to 15. All animals treated with the test substance showed reduced food intake when compared to the control group. Food intake was statistically significantly reduced on gestation days 17 to 20 in the low dose group, days 7 to 10 and 16 to 20 in the mid dose group and days 7 to 22 in the high dose group. The absolute and relative food intake was markedly reduced for high dose females during the whole treatment period. The treatment did not influence drinking water consumption as observed during daily visual appraisal. No test item-related pathological findings were observed for low dose animals. Treatment-related macroscopic changes of the spleen were observed for mid dose females (rough surface of the spleen) and high dose females (rough surface of the spleen and spleen reduced in size). Necropsy of the prematurely deceased dams revealed gastric lesions (stomach: haemorrhagic/dark red foci, ulcers or reddening) in all animals and intestinal changes (dark-red contents) in  one of these dams. Further findings such as emaciation, dark-red lungs and a discoloured heart were noted in one or two deceased dams. The gravid uterus weight, the carcass weight and the net weight change from day 6 onwards (carcass weight minus day 6 body weight) of the animals treated with 300 or 1000 mg/kg bw/day were not influenced. At 3000 mg/kg bw/day, a severe reduction was noted for the gravid uterus weight caused by the  low number of foetuses. The effects seen were attributed to the massive dose of free acid by gavage, an effect that is not seen in studies where the substance is administered via the diet.

Effect levels (maternal animals)

1

Key result

Dose descriptor:

LOAEL

Effect level:

>= 1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

body weight and weight gain

dead fetuses

food consumption and compound intake

pre and post implantation loss

2

Key result

Dose descriptor:

NOAEL

Effect level:

ca. 300 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

body weight and weight gain

gross pathology

Results (fetuses)

Reduction in number of live offspring:

effects observed, treatment-related

Description (incidence and severity):

74.9% reduction in 3000 mg/kg bw/day group.

Changes in sex ratio:

effects observed, non-treatment-related

Changes in litter size and weights:

effects observed, treatment-related

Description (incidence and severity):

Significant reduction in litter size and weights in 3000 mg/kg bw/day group.

Changes in postnatal survival:

effects observed, non-treatment-related

External malformations:

effects observed, treatment-related

Description (incidence and severity):

At 3000 mg/kg b.w./day, 5 malformed fetuses with malrotated fore paws (in 4 fetuses) or
omphalocele (in one fetus) were noted, this incidence of malformations was statistically
significantly (at p ~ 0.01) different from the control. External examination revealed one malformed fetus and one malformed implant (regarded as a late resorption) with malrotated paws at 1 000 mg/kg b. w ./day. This malformation is known to occur spontaneously in this rabbit strain as supported by the historical/background data from the lab.

Skeletal malformations:

no effects observed

Visceral malformations:

no effects observed

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:yes. Remark: Considered to be secondary to maternal gastro-intestinal damage from sorbic acid administration.

Details on embryotoxic / teratogenic effects:
No teratogenic properties up to a dose level of 1000 mg/kg bw/day.
No treatment related effects on the number of corpora lutea, implantation sites, resorptions
or life implantation losses foetuses or on the values calculated for the pre- and post- were observed for low and mid dose females.
Increased post- implantation loss, significant increased total resorptions and accordingly lower number of foetuses occurred at 3000 mg/kg bw/day. The sex ratio of the foetuses was comparable for all tested groups. Treatment at 1000 or 3000 mg/kg bw/day induced significantly decreased mean placental weights and foetal weights. At 3000 mg/kg bw/day, only 5 of 69 foetuses survived the 24 hour incubator stay, which was regarded as test item-related. No test item-related differences between foetuses of the control and low dose groups concerning external variations and malformations, skeletal malformations, variations, retardations and soft tissue examinations were observed. At 3000 mg/kg bw/day, a significant increase in external malformations (mal- rotated right fore paw) was noted. In addition, treatment- related increase in external variations (domed foetal head) was observed at this dose level. Further, a marginal but statistically significant increase in the foetal incidence of accessory 13th ribs was recorded in the high dose group. Significant increases in skeletal retardations included missing ossification of lumbar vertebral bodies, incomplete ossification of the skull, and missing ossification of the talus. At 1000 mg/kg bw/day, a marginal but statistically significant increase was noted for the foetal incidence of missing lumbar vertebral bodies. Soft tissue examinations of the head of high dose foetuses revealed a statistically significant increase of dilated lateral cerebral ventricles.

Effect levels (fetuses)

Key result

Dose descriptor:

NOAEL

Effect level:

>= 1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

external malformations

Fetal abnormalities

Key result

Abnormalities:

no effects observed

Overall developmental toxicity

Key result

Developmental effects observed:

no

Applicant's summary and conclusion

Conclusions:

No teratogenic properties were observed up to a dose level of 1000 mg/kg bw/day.
-- NO(A)EL maternal toxic effects: 1000 mg/kg b.w./day
- NO(A)EL embryotoxic/ teratogenic effects: 1000 mg/kg b.w./day
- NO(A)EL embryotoxic/ teratogenic effects: 300 mg/kg b.w./day

No treatment-related maternal or developmental effects were observed at 300 mg/kg bw/day. Maternal findings in the mid dose group included increased respiratory rate following administration, decreased body weight gain and rough surface of the spleen. Maternal findings in high dose females included increased respiratory rate following administration, death, abortion, decreased body weight and body weight gain, marked decrease in food consumption and pathological findings upon necropsy (rough surface and reduced size of the spleen). Statistically significant reductions in mean foetal and placental weights and the viability of the foetuses were observed at the mid and high dose levels. At 1000 mg/kg bw/day, marginal statistically significantly increased incidences of unclassified macroscopic variations (abdominal distension caused by an inflated gastric tract) and skeletal variations (less than 7 lumber vertebral bodies ossified) occurred. Abdominal distension was noted in two dams where all foetuses were affected and was regarded as not test item-related. At the high dose level, causing severe maternal toxicity, increased post-implantation loss, severely reduced viability of foetuses, increased incidences of malrotated fore paws, domed head, accessory 13th ribs, skeletal retardations according to Dawson and soft tissue variations of the head according to Wilson were recorded. However, it did not appear justified to draw any valid conclusion on teratogenic properties at the highest dose level of this study. Slight or severe maternal toxicity observed at the mid and high dose level, respectively, and severely reduced food consumption at both dose levels indicated malnutrition. This normally results in inadequate intake of calcium and micronutrients like trace elements and vitamins, which are required for normal development of the foetuses. The malnutrition resulted in premature death of dams, retarded development of the foetuses and reduced viability of foetuses in the highest dose group. As a reason, it was considered that sorbic acid was administered over the test period by single gastric intubations per day. Necropsy revealed gastric lesions in all deceased animals. Since sorbic acid is known to have irritant properties, such lesions are probably attributable to administration of a large quantity of the irritant test article by gastric intubation. Furthermore, it cannot be excluded that administration of large quantities of an antimicrobial substance resulted in disturbance of the intestinal microflora which, in turn, would result in deficiencies in nutrients, in particular for rabbits species. It should, in contrast, be noted that in feeding studies for rodents and dogs (with human-like intestinal function), high doses of sorbic acid are well tolerated, which supports these conclusions. Finally, it is noted that The extrapolation from sorbic acid to potassium sorbate or vice versa is considered not to be restricted in any way, since the determinant of potential toxicity is on the "sorbate" anion.

Executive summary:

In conclusion, the test item sorbic acid possessed no teratogenic properties up to
a dose level of 1000 mg/kg b.w./day. Slightly increased incidences of external
malformations were only noted at a materno-lethal dose level of 3000 mg/kg
b.w./day.