Cinnamaldehyde

Endpoint summary

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Effects on fertility

Description of key information

Reproductive Toxicity Study:

The read-across chemical cinnamyl alcohol [CAS 104-54-1; EC: 203-212-3] was given by oral gavage to 12 rats per sex per dose level at 0 (vehicle), 87.5, 175 and 350 mg/kg bw/day. The study was performed according to the OECD 421 (adopted in 2016) and GLP. No adverse effect was observed.The study-derived NOAEL for general, reproductive, and developmental toxicity was 350 mg/kg bw/day.

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

The study contains experimental data of a read-across analogue.

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

In-house bred.

Sex:

male/female

Details on test animals or test system and environmental conditions:

The rats were 9 weeks of age at receipt. Females were nulliparous and non-pregnant.
Body weight at receipt :
Males: 250.25 to 297.51 g
Females: 200.85 to 249.90 g
Animals were housed under standard laboratory conditions in an environmentally monitored, air-conditioned room with adequate fresh air supply (12 to 15 air changes per hour), room temperature 19.6 to 23.2oC and relative humidity 46 to 65%, with 12 hours fluorescent light and 12 hours dark cycle. The temperature and relative humidity were recorded once daily
Animals were housed in a standard polypropylene cage (size: L 430 x B 285 x H 150 mm) with stainless steel mesh top grill having facilities for holding pelleted food and drinking water in water bottle fitted with stainless steel sipper tube. Clean sterilized paddy husk was provided as bedding material.
- During acclimatization, two animals of same sex were housed.
- Pre-mating – In each cage, two animals of the same sex and group were housed.
- Cohabitation Period (mating) – In each cage, two animals (one male and one female) of the same group were housed.
- Post-mating - After confirming presence of sperm in the vaginal smear and/or vaginal plugs (Day 0 of pregnancy), the mated pairs were separated. Males were housed with their former cage mates while females were housed individually. Sterilized paper shreds were provided as a nesting material for mated females from gestation day 20 onwards.

Altromin maintenance diet for rats and mice (manufactured by Altromin Spezialfutter GmbH & Co.KG) was available ad libitum to the animals throughout the experimental period. The contaminant analysis test report of the feed is included as Annexure 2. A sample of feed from the batch used in the study was retained until the finalization of study report and was discarded on the day of finalization of study report.

Water was available ad libitum throughout the acclimatization and experimental period. Deep bore-well water passed through reverse osmosis unit was provided in plastic water bottles with stainless steel sipper tubes.

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

The test item/vehicle was administered by oral (gavage) route using stainless steel intubation cannula attached to a disposable syringe. All the doses were administered in an equivolume of 5 mL/kg with the concentration of 17.5, 35 and 70 mg/mL for low, mid and high dose groups, respectively. Vehicle was administered to the control group at an equivolume of 5 mL/kg body weight. The actual dose volume for each animal was calculated based on the most recent body weight. The test item formulations were administered as soon as possible after preparation

Details on mating procedure:

The males and females were placed in 1:1 ratio. Every morning, the vaginal smear of each female was examined for presence of sperm in the vaginal smear. Each femalerat was housed with its respective male rat until pregnancy occured by evidence of sperm in the vaginal smear or until two weeks. Day ‘0’ pregnancy was confirmed by the presence of sperm in the vaginal smear. Four females one each from groups G1, G2 and two from group G4 were not mated during the 14 days cohabitation period. These females were paired with a proven male on the day 14 of cohabitation till day 17 for animals from groups G1, G2 and till day 15 for the animal from group G4. The females confirmed with mating but not littered were sacrificed on day 25 after gestation day ‘0’.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Homogeneity and dose formulation analysis was done by Analytical Chemistry department of Bioneeds India Private Limited. The analysis was done as per methods detailed in the Study Plan No. BIO-ANM 1588 and the results are presented in the report. Sampling and analysis of the formulations were performed during week 1 and week 5 of the treatment. The samples were collected in duplicates from top, middle and bottom layers from low, mid and high dose concentrations and in duplicates from single layer from vehicle control. The exact volumes of test item formulation samples are included in the study report. The prepared test item formulations were stirred using magnetic stirrer during sampling.The collected samples were transferred to Analytical Chemistry Department of Bioneeds India Private Limited for dose formulation analysis. One set of aliquots of each formulation were analyzed. The second aliquot was stored for backup purpose at established stability conditions. The second set of samples were discarded as the analysis results of first set of samples were within the limits. Formulations were considered acceptable, since the mean results were within the range of 85 to 115% of the nominal concentration and the relative standard deviation (% RSD) is ≤10%.

Duration of treatment / exposure:

The males were treated for two weeks pre-mating, during mating and up to the day before sacrifice during post-mating period (total of 35 days of treatment). Thefemales were treated for two weeks pre-mating period, during mating and pregnancy (gestation) and up to lactation day 13 after which the pups were sacrificed on PND 13 and females (dams) were sacrificed on lactation day 14 after overnight fasting (water allowed).

Frequency of treatment:

Once daily.

Remarks:

0 mg/kg bw/day (vehicle) (G1), 87.5 mg/kg bw/day (G2), 175 mg/kg bw/day (G3), and 350 mg/kg bw/day (G4)

No. of animals per sex per dose:

12

Control animals:

yes, concurrent vehicle

Positive control:

Not included.

Parental animals: Observations and examinations:

Clinical signs, body weight, food intake, gestation length, mating and index fertility.

Oestrous cyclicity (parental animals):

Oestrus cyclicity was monitored for two weeks after the five days of acclimatization to evaluate the normal oestrus cycle (4 to 5 days). Only females with normal oestrus cyclicity were selected for the treatment. Vaginal smears were monitored daily from the beginning of the treatment period until evidence of mating. When obtaining vaginal/cervical cells, care was taken to avoid disturbance of mucosa, which may induce pseudopregnancy. The status of oestrus cyclicity of females was determined on termination day (lactation day 14).

Litter observations:

Number of pups per litter, sex ratio, live birth index, pup survival, pup weight, anogenital distance, male pup nipple/areolae retention.

Postmortem examinations (parental animals):

Uteri observations, hormone levels, organ weight, gross pathology and histopathology as per OECD 421 (2016).

Postmortem examinations (offspring):

Gross pathology

Statistics:

Parametric (one-way ANOVA with Dunnett's post test), Non-parametric (Kruskal-Wallis) and the Chi-square test were used to statistically evaluate the data. P below 0.05 was considered to be statistically significant.

Reproductive indices:

Gestation index, mating index, fertility index, and pregnancy index.

Offspring viability indices:

Live birth index and pup survival index.

Clinical signs:

no effects observed

Mortality:

no mortality observed

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:

no effects observed

Description (incidence and severity):

No significant changes in serum thyroxine (T4) hormone levels were observed in either the adult animals or pups.

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

Microscopic findings were restricted to minimal degeneration of seminiferous tubules in testes observed in two males treated at 350 mg/kg. This finding was considered incidental as the changes were of minimal severity, distributed in focal areas, and unilateral in nature, and as such changes are commonly observed in laboratory rats.

Histopathological findings: neoplastic:

no effects observed

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Description (incidence and severity):

Male mating and fertility indexes were 92, 92, 100, and 83% at 0, 87.5, 175, and 350 mg/kg, respectively. Female mating index was 100% at all dose levels whereas female fertility indexes were 100, 92, 92, and 92% at 0, 87.5, 175, and 350 mg/kg, respectively.

Dose descriptor:

NOAEL

Effect level:

>= 350 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

mortality

body weight and weight gain

food consumption and compound intake

clinical biochemistry

organ weights and organ / body weight ratios

gross pathology

histopathology: non-neoplastic

histopathology: neoplastic

reproductive function (oestrous cycle)

reproductive performance

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

not examined

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:

no effects observed

Description (incidence and severity):

No significant changes in serum thyroxine (T4) hormone levels were observed in either the adult animals or pups.

Urinalysis findings:

not examined

Sexual maturation:

no effects observed

Anogenital distance (AGD):

no effects observed

Nipple retention in male pups:

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Description (incidence and severity):

No gross pathological changes were observed in any of the adult animals or pups.

Histopathological findings:

not examined

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

>= 350 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

viability

sexual maturation

clinical signs

mortality

body weight and weight gain

clinical biochemistry

gross pathology

Reproductive effects observed:

no

Conclusions:

The study-derived NOAEL for general, reproductive, and developmental toxicity was 350 mg/kg bw/day.

Executive summary:

The read-across chemical cinnamyl alcohol [CAS 104-54-1; EC: 203-212-3] was given by oral gavage to 12 rats per sex per dose level at 0 (vehicle), 87.5, 175 and 350 mg/kg bw/day. The study was performed according to the OECD 421 (adopted in 2016) and GLP. Dose levels were selected based largely on the results of an OECD 414 study performed with cinnamaldehyde [CAS 104-55-2; EC: 203-213-9] in rats. In this study, treatment at 500 mg/kg resulted in mortality; significant clinical signs of toxicity such as hypothermia, lethargy, and (or) prostration that were observed in all animals from GD 7 and onwards; lower maternal body weights in GD 17 and 20; significant decreases in food intake; and several gross/histopathology findings. At 250 mg/kg, only a low incidence (8%) of gross stomach pathological findings were observed; hence, 350 mg/kg bw/day was selected as top dose.Results:All animals survived to planned death and there were no clinical signs of toxicity at any dose level. No significant changes in body weight, body weight gain, or food intake were observed. No significant changes in oestrous cyclicity were observed at any dose level. Male mating and fertility indexes were 92, 92, 100, and 83% at 0, 87.5, 175, and 350 mg/kg, respectively. Female mating index was 100% at all dose levels whereas female fertility indexes were 100, 92, 92, and 92% at 0, 87.5, 175, and 350 mg/kg, respectively. No significant changes in pre-coital interval or gestation length were observed. No significant changes in number of implantations, number of post-implantation loss, or number of post-natal loss were observed. All pregnant females delivered live pups and average litter sizes were 11.3, 10.8, 10.8, and 11.1 at 0, 87.5, 175, and 350 mg/kg, respectively. No significant changes in number of live/dead pups born, live birth index, pup weight, sex ratio, or pup viability were observed. At necropsy, no significant changes in absolute or relative organ weight were observed. All pups were normal externally and there were no cases of abnormal behaviour. No significant changes in mean anogenital distance were observed and there were no cases of nipple retention among males. No gross pathological changes were observed in any of the adult animals or pups. Microscopic findings were restricted to minimal degeneration of seminiferous tubules in testes observed in two males treated at 350 mg/kg. This finding was considered incidental as the changes were of minimal severity, distributed in focal areas, and unilateral in nature, and as such changes are commonly observed in laboratory rats. No significant changes in serum thyroxine (T4) hormone levels were observed in either the adult animals or pups.Conclusion:The study-derived NOAEL for general, reproductive, and developmental toxicity was 350 mg/kg bw/day.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

350 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

Data is from a Klimisch 1 datasource and provides a robust study summary.

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

Study 1:

The read-across chemical cinnamyl alcohol [CAS 104-54-1; EC: 203-212-3] was given by oral gavage to 12 rats per sex per dose level at 0 (vehicle), 87.5, 175 and 350 mg/kg bw/day. The study was performed according to the OECD 421 (adopted in 2016) and GLP. Dose levels were selected based largely on the results of an OECD 414 study performed with cinnamaldehyde [CAS 104-55-2; EC: 203-213-9] in rats. In this study, treatment at 500 mg/kg resulted in mortality; significant clinical signs of toxicity such as hypothermia, lethargy, and (or) prostration that were observed in all animals from GD 7 and onwards; lower maternal body weights in GD 17 and 20; significant decreases in food intake; and several gross/histopathology findings. At 250 mg/kg, only a low incidence (8%) of gross stomach pathological findings were observed; hence, 350 mg/kg bw/day was selected as top dose.Results:All animals survived to planned death and there were no clinical signs of toxicity at any dose level. No significant changes in body weight, body weight gain, or food intake were observed. No significant changes in oestrous cyclicity were observed at any dose level. Male mating and fertility indexes were 92, 92, 100, and 83% at 0, 87.5, 175, and 350 mg/kg, respectively. Female mating index was 100% at all dose levels whereas female fertility indexes were 100, 92, 92, and 92% at 0, 87.5, 175, and 350 mg/kg, respectively. No significant changes in pre-coital interval or gestation length were observed. No significant changes in number of implantations, number of post-implantation loss, or number of post-natal loss were observed. All pregnant females delivered live pups and average litter sizes were 11.3, 10.8, 10.8, and 11.1 at 0, 87.5, 175, and 350 mg/kg, respectively. No significant changes in number of live/dead pups born, live birth index, pup weight, sex ratio, or pup viability were observed. At necropsy, no significant changes in absolute or relative organ weight were observed. All pups were normal externally and there were no cases of abnormal behaviour. No significant changes in mean anogenital distance were observed and there were no cases of nipple retention among males. No gross pathological changes were observed in any of the adult animals or pups. Microscopic findings were restricted to minimal degeneration of seminiferous tubules in testes observed in two males treated at 350 mg/kg. This finding was considered incidental as the changes were of minimal severity, distributed in focal areas, and unilateral in nature, and as such changes are commonly observed in laboratory rats. No significant changes in serum thyroxine (T4) hormone levels were observed in either the adult animals or pups.Conclusion:The study-derived NOAEL for general, reproductive, and developmental toxicity was 350 mg/kg bw/day.

Effects on developmental toxicity

Description of key information

Developmental Toxicity Study:

The substance (CAS 104 -55 -2) was given by oral gavage to 25 pregnant Wistar rats at 0 (vehicle), 125, 250 and 500 mg/kg bw/day from GD 5 to 19. The study was performed according to OECD 414 (adopted in 2018) and GLP.NOAEL for maternal systemic toxicity was considered at 250 mg/kg bw/day. This effect level was based on mortality, clinical signs of toxicity, statistically/biologically significant decreases in body weight on GD 17 and 20, significant decreases in food intake on GD 8 and 11, and several gross/histopathology findings. NOAEL for developmental toxicity was considered at 250 mg/kg bw/day. This effect level was based on lower foetal body weights at 500 mg/kg bw/day.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

250 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

Data is from a Klimisch 1 data source and provides a robust study summary.

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

Study 1:

The substance (CAS 104 -55 -2) was given by oral gavage to 25 pregnant Wistar rats at 0 (vehicle), 250, 500 and 1000 mg/kg bw/day from GD 5 to 19. Due to unexpected mortality at 1000 mg/kg, an additional group of 25 pregnant Wistar rats treated at 125 mg/kg bw/day from GD 5 to 19 was included. The study was performed according to OECD 414 (adopted in 2018) and GLP.Results (adults):All animals treated at 0, 125, and 250 mg/kg survived to planned death. At 500 mg/kg, one animal was found dead on GD 9. No clinical signs of toxicity were observed at 125 or 250 mg/kg. At 500 mg/kg, at least two of the following symptoms were observed in all animals from GD 7 and onwards: hypothermia, lethargy, prostration, and excessive salivation.No significant changes in body weight or body weight gain were observed at 125 or 250 mg/kg. At 500 mg/kg, a significant decrease in maternal body weight (by 6.4%) was found on GD 17 as compared to the control group. On GD 20, a trend of lower maternal body weight (by 6.9%) was found at 500 mg/kg as compared to the control group. These body weight effects were preceded bysignificant decreases in maternal food intake at 500 mg/kg on GD 8 (by 31.2%) and on GD 11 (by 13.9%) as compared to the control group.No significant changes in T3, T4 or TSH levels were observed.Pregnancy rates at termination were 72% (18/25), 96% (24/25), 68% (17/25), and 54% (13/24) at 0, 125, 250 and 500 mg/kg, respectively. For pregnant animals, no significant changes in the numbers of corpora lutea, implantation sites, resorptions, pre-implantation loss, or post-implantation loss were observed. No significant changes in absolute or relative weight of the uterus, ovary, or thyroid and parathyroid were observed. No remarkable effects on placenta weight were observed.No gross findings were made at 0 or 125 mg/kg. At 250 mg/kg, two non-pregnant animals showed pathological alterations in stomach which included white spots of muscular portion, increased size/swelling of muscular portions, and swelling of glandular portion. At 500 mg/kg, a total of 15 pregnant/non-pregnant animals showed pathological alterations in stomach which included white spots of muscular tissue, increased size of muscular portion, and increased size/swelling of glandular portion.Microscopic findings at 250 mg/kg included focal hyperkeratosis in stomach in one animal (minimal in severity). At 500 mg/kg, microscopic findings included focal/multifocal hyperkeratosis in stomach; focal alveolar haemorrhage; diffuse degeneration of glandular tissue in stomach; focal erosion of muscular tissue in stomach; leukocyte infiltration in muscular tissue in stomach; and oedema of muscular tissue in stomach.Results (foetuses) :No significant changes in litter size or sex ratio were observed.At 500 mg/kg, a significant decrease in female foetal weight (by 8.2%) and a significant decrease in sex-combined foetal weight (by 6.4%) were observed as compared to the control group. No other significant changes in foetal weight were observed. No significant differences in anogenital distance were observed.Gross foetal findings occurred at low incidences at all dose levels and were not attributed to the test item. No variations or malformations were observed during visceral and head razor examinations. A number of skeletal variations/malformations were observed, however none of them were attributed to the test chemical since they lacked dose dependency and (or) were considered common to developing foetuses.Conclusion:NOAEL for maternal systemic toxicity was considered at 250 mg/kg bw/day. This effect level was based on mortality, clinical signs of toxicity, statistically/biologically significant decreases in body weight on GD 17 and 20, significant decreases in food intake on GD 8 and 11, and several gross/histopathology findings. NOAEL for developmental toxicity was considered at 250 mg/kg bw/day. This effect level was based on lower foetal body weights at 500 mg/kg bw/day

Justification for classification or non-classification

As per CLP guidance, a comparison between the severity of the effects on fertility and (or) development and the severity of other toxicological findings must made during classification. In the above OECD 414 study, the only observed effect on development was a slight decrease in foetal body weight at the top dose level (by 6 to 8% vs. controls) and this occurred in the presence of marked systemic toxicity as evident by mortality, significant clinical signs of toxicity, lower maternal body weights, reduced food intake, and gross/histopathology findings. The amount of maternal body weight reduction at the top dose level (by 6 to 7% vs. controls) was comparable to the amount of foetal body weight reduction and it occurred during late gestation, i.e. on GD 17 and 20. Considering this and that the maternal body weight effect was accompanied by reduced food intake, it is not unreasonable to assume that the lower foetal body weights at 500 mg/kg in the study were secondary or partially secondary to maternal undernutrition (1). By also considering other toxic effects at 500 mg/kg, such as mortality and significant clinical signs of toxicity in all animals from GD 7, the slight decrease in foetal weight at 500 mg/kg was considered to secondary to maternal systemic toxicity. Overall, considering the results of the OECD 414 study with cinnamaldehyde and the OECD 421 study with cinnamyl alcohol, the registered substance is regarded to be classified as Not Classified for reproductive toxicity according to Regulation EC 1272/2008 without further testing. A read-across justification has been attached in Section 13 ("Assessment reports") in IUCLID.

1. Chernoff et al.The relationship of maternal and fetal toxicity in developmental toxicology bioassays with notes on the biological significance of the "no observed adverse effect level". Reprod Toxicol 2008; 25: 192-202.