Potassium (E,E)-hexa-2,4-dienoate

Toxicity to reproduction

Administrative data

Endpoint:

extended one-generation reproductive toxicity - with F2 generation (Cohorts 1A, and 1B with extension)

Type of information:

experimental study

Remarks:

a study requested under the food additives reevaluation commisioned by EFSA

Adequacy of study:

key study

Study period:

2016-2017

Reliability:

1 (reliable without restriction)

Justification for type of information:

Since the sorbate anion in the (eco-) toxicologically relevant moiety and its presence in dissociated or non-dissociated form is entirely determined by pH, extrapolation to potassium sorbate, the substance in question, is not considered to be restricted.

Based on the re-evaluation of sorbic acid (E 200) and potassium sorbate (E 202) by the EFSA Scientific Panel, an extended one-generation reproductive toxicity study (EOGRTS) in rats including the second generation was recomeded in order to reconsider the ADI (EFSA Journal 2015;13(6):4144).

Celanese contacted ECHA on May 14, 2016 (Ref. No INC000000160261) to understand how to appropriately address the OECD 443 study requested by EFSA (i.e. under a different regulatory regime) in the REACH dossier.

ECHA confirmed on May 24, 2016 that we could conduct the study as requested by EFSA under the food law and its only advice was to update our REACH dossier in accordance with Article 22 of REACH. (please see attachment on the ECHA's response)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 443 (Extended One-Generation Reproductive Toxicity Study)

Version / remarks:

an extended one-generation reproductive toxicity study in rats including the second generation

GLP compliance:

yes (incl. QA statement)

Remarks:

dated 23 sept 2018

Justification for study design:

Based on the re-evaluation of sorbic acid (E 200) and potassium sorbate (E 202) by the EFSA, the Scientific Panel recommended an extended one-generation reproductive toxicity study (EOGRTS) according to the OECD 443 guidelines in rats including the second generation (EFSA Journal 2015;13(6):4144).

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

Details on test material:

specific details provided in the study summary

Specific details on test material used for the study:

sorbic acid
CAS NO 110-44-1
appaereance: white powder
Supplier: Sponsor
Batch Number: 0000806305
Expired sate: 25 Dec 2018
Purity: 100%

Test animals

Species:

rat

Strain:

other: Crl:CD (SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

110 males and 110 females, spare animals were removed from the study room after treatment commenced.
males and females unrelated
duration for acclimatazation for P animals was five days before commencement of treatment
Age range of the P animals at the start treatment: males 76 to 82 days old & females 68 to 75 days old
Weight range of the P animlas at the start of the study: males 362 to 485 g & females 212 to 285 g
Temperature and relative humidity: Monitored and maintained within the range of 20-24°C and 40-70%; there were no deviations from these ranges.
Lighting: Artificial lighting, 12 hours light : 12 hours dark.

Administration / exposure

Route of administration:

oral: feed

Details on exposure:

the test substance was incorporated into the diet and the mixture was made up to the weight of the premix with plain diet. The doubling up process was repeated until half of the premix diet was added. then, this premix was diluted with furtehr quatities of plain diet using the doubling up process to prepare test mixes of 15000, 30000 and 60000 ppm.

Details on mating procedure:

P pairing commenced after two weeks of treatment
F1B pairing commenced 10 weeks after seletcion
Male/Female 1:1 from within the same treatment groups (sibling pairing was not permitted)
Duriation of pairing: up to two weeks
Daily check for evidence of mating: ejected copulation plugs in cage tray.
Day 0 of gestation: When positive evidence of mating was detected.
Male/Female separation: Day when mating evidence was detected.
Pre-coital interval: Calculated for each female as the time between first pairing and evidence of mating.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The mean concentrations of sorbic acid in test formulations analyzed during the study were within 4%, confirming the accuracy of formulation and the difference from mean values were within 1%, confirming precise analysis. The procedural recoveries remained within the validated range, confirming the continued robustness of the method.

Duration of treatment / exposure:

Experimenatl start date (pre-study chemistry) 28 July 2016
Experimental completion date: 24 July 2017
Details presented below in "details on study schedule"

Frequency of treatment:

daily

Details on study schedule:

Study initiation (Study Plan signed by Study Director) 5 July 2016
Experimental start date (pre-study chemistry): 28 July 2016
Animal arrival: 30 November 2016
P treatment commenced: 5 December 2016
P animals paired for mating: 19 December 2016
P necropsy:
Males: 13 February to 16 February 2017
Females: 7 February to 11 February 2017
F1 generation treatment commenced: 9 February 2017
F1B animals paired for mating: 24 April 2017
F1A generation necropsy: 18 April to 21 April 2017
F1B generation necropsy
Males: 12 June to 15 June 2017
Females: 6 June to 11 June 2017
Experimental completion date: 24 July 2017
Study completion date: When the Study Director signs the final report.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 ppm

Remarks:

Control

Doses / concentrations 2

Dose / conc.:

15 000 ppm

Remarks:

Group 2

Doses / concentrations 3

Dose / conc.:

30 000 ppm

Remarks:

Group 3

Doses / concentrations 4

Dose / conc.:

60 000 ppm

Remarks:

Group 4

No. of animals per sex per dose:

Group 1 (control): 25 males / 25 females
Group 2: 25 males / 25 females
Group 3: 25 males ? 25 females
Group 4: 25 males / 25 females

Control animals:

yes, plain diet

Details on study design:

P Generation: males and females mated = gestation / lactation
F1 generation:
Cohort 1A - for tox and repor path males & Cohort 1B - for breeding
Cohort 1B - for breading & Cohort 1A for tox and repro path female
gestating / lactation

Positive control:

n/a

Examinations

Parental animals: Observations and examinations:

Animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment. Cages and cage-trays were inspected daily for evidence of animal ill-health amongst the occupant(s). Any deviation from normal was recorded at the time in respect of nature and severity, date and time of onset, duration and progress of the observed condition, as appropriate. During the acclimatization period, observations of the animals and their cages were recorded at least once per day.
A detailed physical examination was performed on each animal to monitor general health at the following time points:
P and selected F1 generation animals Once each week.
The weight of animals was recorded as follows:
P males Day that treatment commenced (Week 0).
Each week.
Before necropsy.
P females Day that treatment commenced (Week 0).
Each week until mating was detected.
Days 0, 7, 14 and 20 after mating.
Days 1, 4, 7, 14, 21 and 28 post-partum.
Before necropsy.

Oestrous cyclicity (parental animals):

Dry smears: Smears were taken daily for 15 days before pairing, using cotton swabs moistened with saline.
Wet smears: After pairing with the male, daily smearing was continued using pipette lavage, until evidence of mating was observed.
For four days before scheduled termination (nominally Days 25 to 28 post-partum) daily vaginal smears were taken and used to determine the stage of the estrous cycle at termination.

Sperm parameters (parental animals):

The following tests were performed:
Sperm motility -
all groups A sample of sperm was expressed from the vas deferens into prewarmed (target 37oC) medium M199, which contained 0.5% w/v bovine serum albumin (BSA Fraction V). A sample for assessment was taken into a 100 µm depth cannula by capillary action and, where possible, at least 200 sperm per animal analysed using the Hamilton Thorne IVOS Computer Assisted Sperm Analyser (CASA).
Sperm morphology -
Groups 1 and 4: A 200 µL aliquot of the sperm/medium mixture (described above) was diluted with 800 µL of 10% neutral buffered formalin. After staining with nigrosine and eosin an air-dried smear was prepared. Slides were examined by light microscopy for the assessment of sperm morphology. At least 200 sperm were assessed for each male, where possible.
Groups 2 and 3: Fixed samples retained for possible future assessment.
Sperm count -
Groups 1 and 4 : The left cauda epididymis of each male was weighed and then the tunica was removed. The portion obtained was weighed then homogenised for at least 30 seconds in 10 mL of a mixture of 0.9% saline and 0.01% merthiolate (SM). An aliquot of this mixture was added to a pre-prepared IDENT stain tube before being assessed for sperm count using CASA.
Group 2 and 3: Retained frozen for possible future assessment.
Homogenization-resistant spermatids count -
Groups 1 and 4: After removal of the tunica, the left testis of each male homogenised for at least 30 seconds in 25 mL of SM. An aliquot of this mixture was added to a pre-prepared IDENT stain tube before being assessed for homogenisation resistant spermatid count using CASA.
Group 2 and 3: Retained frozen for possible future assessment.

Litter observations:

Clinical observations: All litters were examined at approximately 24 hours after birth (Day 1 of age) and then daily thereafter.
Offspring identification: On Day 1 of age each offspring was numbered individually within each litter using a toe tattoo.
Litter size: Daily records were maintained of mortality and consequent changes in litter size from Days 1-21 of age.
On Day 4 of age, litters containing more than ten offspring were reduced to ten by random culling, leaving, whenever possible, five male and five female offspring in each litter; surplus offspring subject to macroscopic examination.
Sex ratio of each litter: Recorded on Days 1, 4 (before and after culling) and on Day 21 of age.
Individual offspring body weights: Recorded on Days 1, 4 (before culling), 7, 14, 21 and 25 of age.
Weaning of offspring: The dam was removed from the litter cage and offspring were weaned on Day 21 of age.

Postmortem examinations (parental animals):

P adult animals:Females - Day 28 postpartum
Males - After 10 weeks of treatment (following weaning of the F1 animals and confirmation that no further mating required).
All adult animals were subject to a complete macroscopic examination. For males, samples for sperm analysis (as detailed below) were taken as soon as possible after death.

Postmortem examinations (offspring):

F1A animals: At approximately 13 weeks of age.
F1B animals: At approximately 20 weeks of age; females on Day 21 of lactation
F1B female that failed to mate: Group 1 female no 457 - an estrous smear was seen following completion of the pairing period and this females was sacrificed as soon as logistically possible.
F1B females failing to produce a viable litter: Day 25 after mating.
All adult animals were subject to a complete macroscopic examination. For males, samples for sperm analysis (as detailed below) were taken as soon as possible after death. For all P and F1B females, the following was recorded:
Uterus: Number of implantation sites.
F1/F2 Generation Offspring (Unselected)
Culls: Day 4 of age (F1/F2)
Scheduled sacrifice: Day 22 of age (F1) Day 21 of age (F2)
Sequence of sacrifice: To allow satisfactory inter-group comparison.
Offspring were subject to a complete macroscopic examination. Additionally, the following procedures were applicable:
Premature deaths (before weaning): Missing offspring and any grossly autolyzed or grossly cannibalized could not be examined. All other remaining offspring dying before weaning were examined; where possible the examination also included an assessment for the presence of milk in the stomach.
The organs weighed, tissue samples fixed and sections examined microscopically for histopathological examination

Statistics:

Parametric analysis was performed if Bartlett's test (1937) for variance homogeneity (p<1%), when p<0.05, comparisons using t-tests, with the error mean square from the one-way analysis of variance. Non-parametric analysis was performed if Bartlett's test was significant at 1% following both logarithmic and squareroot transformations. Kruskal-Wallis’ test (Kruskal and Wallis 1952, 1953) was used to test any differences intergroup. If this was significant (p<0.05), comparisons using Wilcoxon (1945) rank sum tests; for all other comparisons the H1 approximate test was applied. Clinical pathology, litter size and survival indices anogenital distance, sexual maturation and ovarian follicle counts and corpora lutea data, if 75% data were the same value c, Fisher’s exact tests (1973) were performed. Treated groups were compared using pairwise comparisons of e/dose group against the control both for i) values c, as applicable. Sex ratio were analysed by generalised mixed linear model with binomial errors, a logit link function and litter as a random effect (Lipsitz et al 1991). E/treated group was compared to control using a Wald chi-square test. For anogenital distance data, analysis of covariance was performed using the average pup body weight for e/litter as the covariate (Angervall and Carlstrom, 1963). Vaginal opening to first estrus, an exact one-tailed (uppertail) Linear-by-linear test (Cytel 1995) was applied to all groups. For estrous cycles an exact one-tailed (uppertail) Linear-by-linear test (Cytel 1995) was applied to all groups. Gestation length an exact two-tailed Linear-by-linear test (Cytel 1995), with equally spaced scores, was applied. If test was statistically significant (p<0.05), the highest dose-group was excluded and the test reapplied. This ‘step-down’ process was repeated until the test was no longer statistically significant (p≥0.05). .

Reproductive indices:

Mating rate (%)
Conception rate (%)
Fertility index (%)
Gestaion index (%)

Offspring viability indices:

Post implant survival index (%)
Live birth index (%)
Viability index (%)
Lactation index (%)

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One female from the HD was sacrificed for welfare reasons. Macroscopic examination revealed dark abnormal contents in the caecum, rectum and stomach, multiple dark depressions on the forestomach mucosa, pale internal tissue/extremities and red coat staining; however, no microscopic correlations were observed for these gross observations.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Overall bodyweight gains for males at 15000, 30000 or 60000 ppm during Weeks 0 to 10 of treatment was similar to Controls.
Overall body weight gain during the two-week pre-pairing period was significantly lower than Controls for females receiving 30000 or 60000 ppm (p<0.05).
On Day 0 of gestation mean body weight for females at 60000 ppm was significantly lower than Controls (p<0.05). Body weight gain during the first two weeks of gestation was similar across all groups. During Days 14 to 20 weight gain for females at 30000 and 60000 ppm was significantly lower than Controls (p<0.05 and p<0.01, respectively). The overall gain (Days 0 to 20) at 60000 ppm was subsequently low (p<0.05) but not for 30000 ppm.
During Days 1 to 4 of lactation, bodyweight gain for females at 15000, 30000 or 60000 ppm, was like Controls. During Days 4 to 7 bodyweight gain for females at 60000 ppm was slightly low (p<0.05) and from Days 7 to 14 of lactation, mean bodyweight gain was high in all treated groups, with the difference attaining statistical significance at 30000 and 60000 ppm (p<0.05 and p<0.01, respectively).

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

During Week 1 of treatment food consumption for males receiving sorbic acid was significantly lower than Controls (p<0.05) and there was no evidence of a dose-response.
During Week 2 food consumption values for males receiving sorbic acid in all groups and for females at 30000 or 60000 ppm were marginally lower than Controls. During gestation females at 60000 ppm showed marginally but statistically low food consumption when compared with Controls (Days 0-6, p<0.05; Days 7-19, p<0.01). From Day 6 of gestation females receiving 30000 ppm showed slight but statistically low food consumption (Days 7-19, p<0.01).
Throughout lactation, food consumption for females at 60000 ppm was significantly lower than Controls (p<0.01). During Days 14-20 of lactation food consumption was low at 15000 and 30000 ppm (p<0.05 and p<0.01, respectively).

Food efficiency:

no effects observed

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

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

All differences were minor and/or restricted to a single sex and considered to be within normal biological variation.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Differences including those that attained statistical significance were minor or lacked a dose response and were not considered to be treatment-related.

Urinalysis findings:

effects observed, non-treatment-related

Description (incidence and severity):

Urinalysis at termination revealed high pH in males and females at 60000 ppm (106 and 109% of Controls, respectively; p<0.01 females only), and low total potassium for females at 60000 ppm (72%; p<0.01) when compared with Controls.
All other differences were considered to be within normal biological variation.

Behaviour (functional findings):

not examined

Immunological findings:

no effects observed

Organ weight findings including organ / body weight ratios:

not specified

Histopathological findings: non-neoplastic:

not specified

Description (incidence and severity):

There were no microscopic findings that could be related to treatment for males and females examined from the parental generation.
Microscopic examination of tissues from Group 2 male no.31 which was considered to have suspect fertility (no motile sperm) revealed seminiferous tubular atrophy, reduced numbers of sperm in the epididymis, and spermatocele granoloma(ta) in the testis and epididymis. There were no microscopic correlates observed for the remaining animals with suspect fertility, namely Group 2 male nos. 26, 31 and 32 and Group 2 female nos. 126, 130, 131, 139, and 143.
Seminiferous tubules were evaluated with respect to their stage in the spermatogenic cycle and the integrity of the various cell types present within the different stages. No cell or stage specific abnormalities were noted.

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Description (incidence and severity):

Thyroid hormone analysis:
Thyroid stimulating hormone (TSH): Differences in TSH levels were observed at each sampling point when compared with the Controls. These differences in TSH levels from Controls were not consistent and were attributed to natural variation rather than dietary administration of sorbic acid.
Thyroxine (T4): There were no differences in the thyroxine levels for adult male or female animals at scheduled sacrifice of either the parental generation, Cohort A of the F1 generation or selected F1 offspring after weaning on Day 22 of age that could be attributed to treatment with sorbic acid.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

Details on results (P0)

Dietary inclusion of sorbic acid at dietary concentrations up to and including 60000 ppm had no effect on clinical condition, estrous cycles, mating performance, fertility, gestation length, gestation index. In addition, there were no treatment related changes in hematology, thyroid-related hormone levels (TSH and T4), sperm parameters, male organ weights, parental macropathology and histopathology in any dose group at scheduled termination.
Effects observed in the groups that received sorbic acid included
•Sporadic differences in body weight, body weight gain and food consumption at 30000 ppm
•Consistently low body weight, bodyweight gain and low food consumption at 60000 ppm.
•High absolute and body weight-relative group mean liver weight for females at 60000 ppm on Day 28 post-partum.

Effect levels (P0)

Key result

Dose descriptor:

NOAEL

Remarks:

Based on the lower food consumption, body weights and body weight gains in all cohorts at the High dose.

Effect level:

>= 3 000 mg/kg bw/day (nominal)

Based on:

test mat.

Remarks:

Sorbic acid 99% purity

Sex:

male/female

Basis for effect level:

body weight and weight gain

food consumption and compound intake

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

Group 3 female no. 489 (30000 ppm; Cohort 1B) was euthanised for welfare reasons on Day 35 of the F1 generation; terminal signs included abdominal distension, loose faeces, dull eyes and pale skin. Macroscopically, this animal had a thickened duodenum, ileum, caecum, and colon and an enlarged spleen which correlated to microscopic mucosal ulceration and inflammatory cell infiltrate in the small and large intestine and extramedullary haemopoiesis in the spleen, respectively. Other microscopic findings included myocardial necrosis in the heart, extramedullary haemopoiesis in the liver, increased apoptotic cells in the thymus, adrenal cortical hypertrophy, increased granulopoiesis in the bone marrow, inflammatory cell infiltrates in the meninges of the brain, the eyes, and the adrenals, sparse corpora lutea in the ovaries, atrophy of the uterus, vagina, and mammary acini, decreased cellularity in the spleen and lymph nodes, and plasmacytosis in the lymph nodes. The major factor that contributed to the early euthanasia of Group 3 female 489 was the gastrointestinal lesion(s). The findings in the heart, liver, spleen, bone marrow, brain, eyes, adrenals, and lymph nodes suggest that the animal may have been septicaemic. The bone marrow finding was likely a compensatory secondary response to the inflammatory cell infiltrates observed in multiple tissues. The findings in the thymus, ovaries, uterus, vagina and mammary were likely stress-induced [Everds et al., 2013]. These isolated findings in the Group 3 female were not considered treatment-related.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

The bodyweight of offspring on Day 1 of age and the subsequent gain up to Day 4 of age was unaffected by parental treatment with sorbic acid. During Days 4-7 of age and Days 14-21 of age body weight gain was low at 60000 ppm resulting in a low absolute body weight for offspring on Day 21 of age. Bodyweight gain from weaning on Day 21 of age up to Day 25 of age remained marginally low for offspring receiving 60000ppm.

Food consumption and compound intake (if feeding study):

not specified

Description (incidence and severity):

Overall (total) food consumption for selected F1 males at 60000 ppm was considered low when compared with Controls. Overall values at 15000 ppm and 30000 ppm were similar to Controls.

Food consumption for selected F1 females at 30000 ppm and 60000 ppm were considered low when compared with Controls; overall values at 15000 ppm were similar to Controls.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

There were no significant toxicological findings in animals at termination for hematological endpoints.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Males and females at 60000 ppm had high cholesterol levels; the difference for males attained statistical significance (p<0.01)
Females at 60000 ppm showed significantly high creatinine levels (p<0.05)
All other differences including those that attained statistical significance were minor or lacked relationship to the dietary concentration.

Urinalysis findings:

effects observed, non-treatment-related

Description (incidence and severity):

After 9 weeks of treatment Cohort A males at 30000 and 60000 ppm showed low urinary output (p<0.01), high specific gravity (p<0.05), low total creatinine (p<0.05) and low total potassium (p<0.05) when compared with Controls; these differences were not evident for males that received 15000 ppm or for females at all dietary concentrations.
Differences for females after 9 weeks of treatment were limited to low total urinary protein at 60000 ppm (p<0.01).

Sexual maturation:

no effects observed

Description (incidence and severity):

The day of age that males completed balano preputial separation was similar across all groups; with the mean bodyweight on completion showing inter group/cohort variation.
Vaginal opening at 60000 ppm was slightly delayed in both cohorts (3 days in Cohort A and 2 days in Cohort B). At 15000 and 30000 ppm the day of age at opening was similar to Controls. The mean bodyweight on completion of vaginal opening was slightly but significantly higher than Controls for Cohort A females (p<0.05). This difference was not evident for Cohort B and is not considered to be toxicologically significant.

Anogenital distance (AGD):

no effects observed

Description (incidence and severity):

The ano-genital distance for F1 offspring on Day 1 of age was unaffected by paternal treatment with sorbic acid.

Nipple retention in male pups:

no effects observed

Description (incidence and severity):

No nipples were observed for F1 males on Day 13 of age.

Organ weight findings including organ / body weight ratios:

not specified

Gross pathological findings:

no effects observed

Description (incidence and severity):

Macroscopic examination of males after 10 weeks of treatment and females on Day 28 post partum did not reveal any findings that could be attributed to dietary administration of sorbic acid.

Histopathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no microscopic findings that could be related to treatment for males and females examined from the parental generation.
Microscopic examination of tissues from Group 2 male no.31 which was considered to have suspect fertility (no motile sperm) revealed seminiferous tubular atrophy, reduced numbers of sperm in the epididymis, and spermatocele granoloma(ta) in the testis and epididymis. There were no microscopic correlates observed for the remaining animals with suspect fertility, namely Group 2 male nos. 26, 31 and 32 and Group 2 female nos. 126, 130, 131, 139, and 143.
Seminiferous tubules were evaluated with respect to their stage in the spermatogenic cycle and the integrity of the various cell types present within the different stages. No cell or stage specific abnormalities were noted.

Other effects:

no effects observed

Description (incidence and severity):

Thyroid hormone analysis:
Thyroid stimulating hormone (TSH): Differences in TSH levels were observed at each sampling point when compared with the Controls. These differences in TSH levels from Controls were not consistent and were attributed to natural variation rather than dietary administration of sorbic acid.
Thyroxine (T4): There were no differences in the thyroxine levels for adult male or female animals at scheduled sacrifice of either the parental generation, Cohort A of the F1 generation or selected F1 offspring after weaning on Day 22 of age that could be attributed to treatment with sorbic acid.

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Effect levels (F1)

Key result

Dose descriptor:

NOAEL

Effect level:

>= 3 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

Results: F2 generation

General toxicity (F2)

Clinical signs:

no effects observed

Mortality / viability:

not specified

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

The bodyweight on Day 1 of age was unaffected by parental treatment with sorbic acid. From Day 1of age up to weaning on Day 21 of age body weight gain was low at 30000 ppm and 60000 ppm resulting in a low mean body weight for offspring at both dose levels on Day 21 of age.

Food consumption and compound intake (if feeding study):

not specified

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

Sexual maturation:

no effects observed

Anogenital distance (AGD):

no effects observed

Nipple retention in male pups:

effects observed, non-treatment-related

Description (incidence and severity):

Limited to group 2 (litter No 476, M2 5) 2 nipple count and group 3 (litter No 486, M3 5) 1 nipple count. thses observations were considered to be unrelated to the parental treatment with sorbic acid.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

The group mean body weight relative heart, liver and spleen weights for Cohort A males that received 60000 ppm were higher than Controls (p<0.05). Group mean body weight relative spleen weight for males at 30000 ppm was also high (p<0.05). These differences were not apparent in males that received 15000 ppm or in Cohort A females at all dietary concentrations up to and including 60000 ppm.
The reproductive organ weights for males from both Cohort A (after 9 weeks of the F1 generation) and Cohort B (after 17 weeks of the F1 generation) were considered to be unaffected by treatment. The minor differences at 60000 ppm were attributed to the effect on body weight rather than a direct effect of treatment.
On Day 21 of lactation females that received 60000 ppm showed low absolute and body weight relative group mean ovarian and uterine weights when compared with Controls (p<0.05/p<0.01).

Gross pathological findings:

no effects observed

Histopathological findings:

no effects observed

Developmental neurotoxicity (F2)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F2)

Developmental immunotoxicity:

not examined

Effect levels (F2)

Key result

Dose descriptor:

NOAEL

Generation:

F2

Effect level:

>= 3 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

Overall reproductive toxicity

Key result

Reproductive effects observed:

no

Treatment related:

no

Applicant's summary and conclusion

Conclusions:

Dietary administration of sorbic acid at levels of 15000, 30000 and 60000 ppm was generally well tolerated by the parental generation, F1 offspring and adults and F2 offspring. There were no treatment-related adverse effects on clinical condition, thyroid-related hormones, estrous cycles, mating performance and fertility, organ weights, clinical chemistry, urinalysis, hematology, immunophenotyping, sperm parameters, macropathology and/or histopathology.
At 30000 ppm there was sporadically lower food consumption, body weight and body weight gain noted in most cohorts. At 60000 ppm lower food consumption, body weight and body weight gain was of greater magnitude and consistently observed in all cohorts.
Differences in F1/F2 offspring body weight gain had no effect on clinical condition or survival. The magnitude of the effect on the F1 offspring at 60000 ppm and the F2 offspring at 30000 ppm was not considered adverse. At 60000 ppm the differences in F2 offspring body weight gain was considered an adverse effect of treatment with sorbic acid.
Due to the consistency and magnitude of lower food consumption, body weights and body weight gains in all cohorts receiving 60000 ppm sorbic acid in the diet, this dietary concentration was considered to be the LOAEL. The no observed adverse effect level
(NOAEL) for systemic toxicity and reproductive performance in the adult parental animals and for offspring is 30000 ppm.

Executive summary:

The objective of this study was to assess the effect of sorbic acid (a food preservative) on systemic toxicity, reproductive performance and development when administered continuously in the diet to Sprague-Dwaley (CD) rats. Three groups of 25 male and 25 female rats received sorbic acid via the diet, at concentrations of 15000, 30000 or 60000 ppm for two weeks before pairing, throughout pairing, gestation, lactation and until termination.  A similarly constituted Control group received untreated basal diet.  For the P generation data were recorded on clinical condition, body weight, food consumption, estrous cycles, mating performance and fertility, gestation length and parturition observations and reproductive performance.  Clinical pathology (hematology, blood chemistry, urinalysis and thyroid-related hormones), sperm assessment, organ weight, macroscopic pathology and microscopic pathology investigations were performed.  

The F1 generation comprised of two cohorts:

Cohort 1A: 20 male and 20 female progeny were selected from each dose group and continued to receive the relevant diet, from approximately mid lactation (when offspring are expected to start to consume diet) until scheduled sacrifice at approximately Week 13 of age.  

Cohort 1B: 20 male and 20 female progeny were selected from each dose group and continued to receive the relevant diet from approximately mid lactation (when offspring were expected to start to consume diet) until scheduled sacrificed following rearing of the F2 offspring to weaning.

Dietary inclusion of sorbic acid at dietary concentrations up to and including 60000 ppm had no effect on clinical condition, estrous cycles, mating performance, fertility, gestation length, gestation index.  In addition, there were no treatment related changes or adverse effects in hematology, thyroid-related hormone levels (TSH and T4), sperm parameters, organ weights, parental macropathology and histopathology in any dose group at scheduled termination.

Effects observed in the groups that received sorbic acid included:

       Sporadic differences in body weight, body weight gain and food consumption at 30000 ppm

       Consistently low body weight, bodyweight gain and low food consumption at 60000 ppm.

F1 Litter

General clinical condition, litter size, offspring survival, sex ratio, ano-genital distance, male nipple count,  thyroid-related hormone levels (TSH and T4) on Day 22 of age, organ weight and macropathology of offspring that were not selected to form the next generation were unaffected by dietary inclusion of sorbic acid at dose levels up to and including 60000 ppm.

The bodyweight of offspring on Day 1 of age and the subsequent gain up to Day 4 of age was unaffected by parental treatment with sorbic acid.  During Days 4-7 of age and Days 14-21 of age body weight gain was low at 60000 ppm resulting in a low absolute body weight for offspring on Day 21 of age.  Bodyweight gain from weaning on Day 21 of age up to Day 25 of age remained marginally low for offspring receiving 60000ppm.

F1 Generation (Cohort A and B)

Dietary inclusion of sorbic acid up to 60000 ppm had no treatment –related changes or adverse effects on clinical condition, estrous cycles, mating performance, fertility, gestation length, gestation index, sexual maturation, hematology, immunophenotyping end points, thyroid-related hormone levels (TSH and T4), sperm parameters, organ weights, parental macropathology, Cohort A ovarian follicle count, corpora lutea count and histopathology. Effects observed in the groups that received sorbic acid included:

       Sporadic differences in body weight, body weight gain and food consumption at 30000 ppm

       Consistently low body weight, bodyweight gain and low food consumption at 60000 ppm.

F2 Litter

General clinical condition, litter size, offspring survival, sex ratio, ano-genital distance, male nipple count and macropathology of offspring were unaffected by dietary inclusion of sorbic acid at dose levels up to and including 60000 ppm.

The bodyweight on Day 1 of age was unaffected by parental treatment with sorbic acid.  From Day 1of age up to weaning on Day 21 of age body weight gain was low at 30000 ppm and 60000 ppm resulting in a low mean body weight for offspring at both dose levels on Day 21 of age.

Differences in F1/F2 offspring body weight gain had no effect on clinical condition or survival.  The magnitude of the effect on the F1 offspring at 60000 ppm and the F2 offspring at 30000 ppm was not considered adverse.  At 60000 ppm the differences in F2 offspring body weight gain was considered an adverse effect of treatment with sorbic acid.

Due to the consistency and magnitude of lower food consumption, body weights and body weight gains in all cohorts receiving 60000 ppm sorbic acid in the diet, this dietary concentration was considered to be the LOAEL.  The no observed adverse effect level (NOAEL) for systemic toxicity and reproductive performance in the adult parental animals and for offspring is 30000 ppm.