Melaleuca alternifolia, ext.

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Effect on fertility: via oral route

Endpoint conclusion:

no study available

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Effects on developmental toxicity

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

- Source: Tweed Ti-Tree Products, PO BOX 1990, Kingscliff 2487, New South Wales, Australia
- Batch No.: A352
- Appearance: Colourless to pale yellow liquid
- Purity: 100%
- Content of terpinen-4-ol: 37%
- Production date: 17 December 2008
- Expiration date of the lot/batch: 17 December 2011
- Storage condition of test material: Room temperature (15-25°C), avoiding direct sunlight, nitrogen headspace.

Species:

rat

Strain:

other: Hannover Wistar

Remarks:

HsdHan:WIST

Details on test animals or test system and environmental conditions:

- Source: WOBE Kft. 1164 Budapest, Garmada u. 10, Hungary
- Age at study initiation: Young adult rats, approximately 16-17 weeks old at onset of study (first start of mating).
- Weight at study initiation: 211-288 g on the day of mating (GD0, all females); 217-288 g at onset of treatment (GD5, evaluated pregnant females; within ± 20% of the mean weight for each sex/group).
- Housing: Prior to and during the mating period: 1-3 animals of the same sex/cage. Mating hours: 1 male and 1-3 females/cage. During pregnancy: 1-4 sperm-positive females/cage.
- Bedding: Lignocel® Hygienic Animal Bedding produced by J. Rettenmaier & Söhne GmbH+Co.KG, D-73494 Rosenberg Holzmühle 1 Germany and Laboratory Animal Bedding produced by Brandenburg Holzfaserstoffe Gmbh& Co.KG, Arkeburger Str. 31, 49424 Goldenstedt, Germany were available to animals during the study.
- Diet: ssniff® SM R/M-Z+H "Autoclavable complete feed for rats and mice – breeding and maintenance" (ssniff Spezialdiäten GmbH Soest, Germany) ad libitum. The food is considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study.
- Water: Tap water from the municipal supply (500 mL water bottle) was available ad libitum. Water quality control analysis was performed every three months and microbiological assessment was performed monthly by Veszprém County Institute of State Public Health and Medical Officer Service (ÁNTSZ, H-8201 Veszprém, József A.u.36., Hungary).
- Acclimation period: At least 6 weeks (staggered mating).
- Temperature: 20.1 - 23.7°C. The temperature was recorded twice daily during the study and the acclimation period. No deviation from the target range occurred.
- Relative humidity: 34 - 69%. The relative humidity was recorded twice daily during the study and the acclimation period. No deviation from the target range occurred.
- Air changes: 15-20 air exchanges per hour.
- Photoperiod: 12 hrs light, 12 hrs dark (artificial light).
- Enrichment: Rodents were group-housed, to allow social interaction; deep wood sawdust bedding was used to allow digging and other normal rodent activities.

Route of administration:

oral: gavage

Vehicle:

other: Polyethylene glycol 400 (PEG 400)

Remarks:

See 'Any other information on materials and methods' for further details.

Details on exposure:

- Justification for vehicle / route of administration: PEG400 was selected as one of the vehicles widely used on general and developmental toxicology studies in the rat, based on previous formulation, analytical and experimental work conducted with this test item. The oral route was considered suitable to provide the systemic exposure required in this developmental toxicology study.
- Concentration in vehicle: 5, 25 and 62.5 mg/mL
- Amount of vehicle: 4 mL/kg bw/day. The volume of the treatment was based on the most recent individual body weight of the animals.
- Lot no.: 1435799

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Each formulation was analysed on 3 occasions to determine the achieved concentration and homogeneity, using a validated UV HPLC method. Duplicate samples were collected for analysis from the top, middle and bottom of each Tea Tree Oil formulation container on 3 occasions during the treatment period. Samples were collected on the first and last week of treatment and at an intermediary time point. For the control group, a single sample was taken for analysis on each sampling occasion for concentration measurements. The samples were analysed immediately after formulation using a validated analytical method (HPLC using UV detection). Tea Tree Oil concentration was determined on the basis of the measured terpinen-4-ol concentration (which was used as an analytical marker).

Details on mating procedure:

- Impregnation procedure: The oestrus cycle of female animals was examined before pairing. After acclimatisation, the females were paired according to their oestrus cycle with males in the morning for up to approximately 3 hours (1 male:1-3 females) until 25, 26, 26 and 27 sperm positive females/group were attained in groups 1, 2, 3, and 4, respectively. After each daily mating period, a vaginal smear was prepared and stained with 1% aqueous methylene blue solution. The smear was examined with a light microscope; the presence of a vaginal plug or sperm in the vaginal smear was considered as evidence of copulation (GD0). Sperm positive females were separated and caged in groups of 1-4 animals.

Duration of treatment / exposure:

From days 5 to 19 of gestation (GD 5 to GD 19)

Frequency of treatment:

Daily treatment by oral gavage 7 days/week, at a similar time each day.

Duration of test:

20 days (GD 0 to GD 20).

Dose / conc.:

0 mg/kg bw/day

Remarks:

Group 1. Control (vehicle only - PEG 400).

Dose / conc.:

20 mg/kg bw/day

Remarks:

Group 2. Low dose.

Dose / conc.:

100 mg/kg bw/day

Remarks:

Group 3. Mid dose.

Dose / conc.:

250 mg/kg bw/day

Remarks:

Group 4. High dose.

No. of animals per sex per dose:

Up to 27 females per dose level were mated in order to achieve a sufficient number of pregnant females (litters for evaluation)/group. See 'Any other information on materials and methods' for further details on experimental design.

Control animals:

yes, concurrent no treatment

Details on study design:

- Justification for dose levels: The dose levels were selected following the conduct of a separate dose range finding study.
- Randomisation: The sperm-positive assumed pregnant females were allocated to each experimental group (on each mating day) in such a way that the group averages of the body weight were as similar as possible. Females paired with the same male were allocated to different groups on the same mating day.

Maternal examinations:

- Mortality and clinical observations: Animals were inspected for signs of morbidity and mortality and clinical observations were made twice daily (at the beginning and end of each working day). Observations were performed on the skin, fur, eyes and mucous membranes, autonomic activity (lachrymation, piloerection, pupil size, respiratory pattern, occurrence of secretions and excretions), circulatory and central nervous system, somatomotor activity and behaviour pattern, changes in gait, posture and response to handling. Particular attention was directed to observation of tremors, convulsions, salivation, diarrhoea, lethargy, sleep and coma. Pertinent behavioural changes and all signs of toxicity including mortality were recorded including onset, degree and duration of signs as applicable. On GD13 and/or 14 the sperm positive females were checked for the presence of vaginal bleeding or “placental sign” (intrauterine extravasation of blood as an early sign of pregnancy in rat, which is considered to confirm implantation).

- Body weight measurement: Individual body weights were recorded on GD 0, 3, 5, 8, 11, 14, 17, and 20 with an accuracy of 1 g.

- Food consumption measurement: The given/remaining food for each cage was weighed on GD 0, 3, 5, 8, 11, 14, 17 and 20 with an accuracy of 1 g. Food consumption was calculated for each measured interval, including for GD 0-20.

- Caesarean section and necropsy procedures: Before the expected delivery, on GD20, surviving females were anesthetized by intraperitoneal injection with pentobarbital sodium (Euthasol 40%), then subjected to caesarean section followed by exsanguination for euthanasia and necropsy. All females, including the ones found dead, were examined macroscopically for any structural abnormalities or pathological changes. Organs with macroscopic alterations were retained in 10% buffered formalin solution for possible microscopic examination and were archived with the study specimens. No histopathological evaluation was performed during this study.
For females surviving to termination on GD20, the corrected body weight was calculated (body weight on GD20 minus weight of the gravid uterus).

Ovaries and uterine content:

The ovaries and uterus were removed and pregnancy status ascertained. The uterus including the cervix was weighed and examined for early and late embryonic or fetal deaths and for the number of live fetuses. When no implantation sites were evident but corpora lutea were present, the uterus was stretched and held in front of a light source to clearly identify the implantation sites. Uteri that appeared non-gravid were further examined to confirm the non-pregnant status by ammonium sulphide staining.

The number of corpora lutea in each ovary and implantation sites in each uterine horn, the number of live fetuses, early and late embryonic death and fetal death was counted and the number and percent of pre and post-implantation losses was calculated. The degree of resorption was described in order to estimate the relative time of death of the conceptus. The placentas were examined macroscopically.

Fetal examinations:

Each live fetus was weighed individually (accuracy 0.01 g) and subjected to external examination. The gender of fetuses was determined according to the anogenital distance. Thereafter the fetuses were individually identified. Approximately half of each litter was subjected to visceral examination and the other half was processed for skeletal examination.

For the fetuses subjected to visceral examination, the abdominal and thoracic region was opened and the thymus and great arteries were freshly examined by means of a dissecting microscope. The rest of the body was fixed in Sanomiya mixture, then after fixation the body was micro dissected by means of a dissecting microscope. The heads were examined by Wilson's free-hand razor blade method.

For the fetuses subjected to skeletal examination, the abdominal region was opened and the viscera and skin of fetuses were removed and the cadaver was fixed in alcian-blue - acetic acid - ethanol mixture. After fixation in isopropanol the skeletons were stained by KOH-Alizarin red-S method and examined using a dissecting microscope.

All abnormalities (external, soft tissue and skeletal malformations, and variations) found during the fetal examinations were recorded.

Statistics:

Data was statistically analyzed using SPSS PC+4.0 software (SPSS Hungary Kft, Budapest). The homogeneity of variance between groups was checked by Bartlett`s homogeneity of variance test. Where no significant heterogeneity was detected, a one-way analysis of variance (ANOVA) was made. If the obtained result was significant Duncan’s Multiple Range test was used to assess the significance of inter-group differences. Significant results with inter-group comparisons were further compared using Kruskal-Wallis and Mann-Whitney U-tests.

The limit for growth retarded fetuses was calculated from the average body weight of the vehicle control fetuses. A fetus was considered as growth retarded if the deviation from the mean control values was greater than minus two fold standard deviation of all control fetuses. Non-pregnant females or females with no implantation or ≤ 5 implantation sites were excluded from the statistical analysis. In-life individual data are presented in the study report as appropriate.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

At 250 mg/kg bw/day, mortality occurred in 7/27 females between GD8 and GD11. Clinical signs prior to death included noisy respiration, decreased activity and/or piloerection were observed in 2/7 animals. No test item related clinical signs were noted in the remaining 5/7 animals. In 16/20 surviving females in the high dose group, clinical signs including decreased activity, hunched back position, noisy respiration, piloerection, red spots on the tail and/or soft feces were observed from GD7 and were considered potentially related to test item administration. No test item related clinical signs were noted in the remaining 4/20 animals.

There were no mortalities in animals treated at 100 mg/kg bw/day, although clinical signs such as noisy respiration, decreased activity, hunched back position, red spots and/or soft feces were noted in 13/26 females.

There were no mortalities in animals treated at 20 mg/kg bw/day. Noisy respiration or soft feces were noted in 3/26 and 1/26 rats, respectively. Since the incidences were low, did not attain statistical significance and generally occurred on single occasions, it is concluded that there were no toxicologically relevant clinical signs at this dose level.

Mortality:

mortality observed, treatment-related

Description (incidence):

No mortalities occurred at 100 or 20 mg/kg bw/day, whereas 7 of the 27 high dose (250 mg/kg bw/day) females were found dead between GD8 and GD11. A specific cause of death was not determined for these animals.

Mortalities seen at the 250 mg/kg bw level indicates that fetal development would have been severely compromised because of the extreme toxicity seen in maternal animals.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Statistically significant reductions in mean body weight were noted in the high and mid-dose groups. Compared with controls over the period of gestation (GD0-20), overall body weight gain in the 100 and 250 mg/kg bw/day groups was -20% and -45%, respectively (p<0.01). There were no effects on body weight in the 20 mg/kg bw/day group that could be attributed to treatment.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

A statistically significant reduction in food consumption was noted in the high and mid-dose groups. Compared with the controls over the period of gestation (GD0-20), mean daily feed intake in the 100 and 250 mg/kg bw/day groups was -12% and -31%, respectively (p<0.01). Tea Tree Oil administered at 20 mg/kg bw/day did not result in statistically significant changes in food consumption.

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

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Statistically lower than control mean gravid uterine weight was noted at 250 mg/kg bw/day (-31%, p<0.01) and lower terminal mean body weight when corrected for gravid uterine weight was noted on GD20 at 100 and 250 mg/kg bw/day (-5%, p<0.05 and -10%, p<0.01, respectively).

The GD20 body weight gain corrected for uterine weight was lower than control at all the dose levels tested when evaluated versus GD0. However, when evaluated versus GD5 (onset of treatment), the GD20 body weight gain corrected for uterine weight was lower than control at 100 and 250 mg/kg bw/day (-58% and -96%, respectively, p<0.01). These adverse effects were considered to be related to Tea Tree Oil administration.

No statistically significant changes were observed at 20 mg/kg bw/day dose level.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Bilateral enlarged adrenals, potentially associated with Tea Tree Oil administration were observed in all 7/7 animals found dead in the high dose group and in 6 of the 20 surviving females. Diffuse discoloration in the lungs of several dead animals was considered to have occurred post-mortem.

There were no macroscopic findings considered related to Tea Tree Oil administration at up to and including 100 mg/kg bw/day, with the exception of one mid-dose female that had bilateral enlarged adrenals.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

Up to 27 females/group were mated to ensure sufficient litters for evaluation. At least 20 litters/group were achieved, with the exception of the high dose group which had 16 evaluable litters on GD20. This was due primarily to maternal mortality and total embryonic loss. The number of litters available for evaluation was sufficient to meet the requirements as stated in OECD 414.

Pre- and post-implantation loss:

effects observed, treatment-related

Description (incidence and severity):

When evaluated on a per group basis, a statistically higher pre-implantation loss occurred at 250 mg/kg bw/day (19 pre-implantation losses versus 11 in the control group, p<0.05). This finding was considered unlikely to be related to treatment, as dose administration started on GD5, after most implantations were complete. A higher number of late embryonic deaths (post-implantation losses) occurred in the high dose group, leading to an overall higher total intrauterine mortality. Pre- and post-implantation losses were unaffected in the low and mid-treatment groups.

When evaluated on a per litter basis, the numbers of corpora lutea and implantation sites were higher in the treated groups when compared to the controls. There were no significant differences in pre-implantation loss between the treated and control animals. Higher values, without statistical significance, were observed for early and late embryonic death, post-implantation loss and total intrauterine mortality at 250 mg/kg bw/day when compared to controls. In view of the adverse effects in the dams, the post-implantation mortality could be considered secondary to maternal toxicity. Other reproductive parameters were unaffected.

Total litter losses by resorption:

not specified

Early or late resorptions:

not specified

Dead fetuses:

no effects observed

Changes in pregnancy duration:

not examined

Description (incidence and severity):

Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): not examined

Changes in number of pregnant:

not examined

Details on maternal toxic effects:

Severe maternal toxicity was seen in animals from the 100 and 250 mg/kg bw/day groups, as evidenced by clinical signs, reductions in food consumption and body weight gain reductions of 20% and 45% respectively between GD0 and GD20.

Dose descriptor:

NOAEL

Effect level:

20 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

clinical signs

Dose descriptor:

NOAEL

Effect level:

20 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

body weight and weight gain

Dose descriptor:

NOAEL

Effect level:

20 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

food consumption and compound intake

Dose descriptor:

NOEL

Effect level:

100 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

mortality

Dose descriptor:

NOAEL

Effect level:

20 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

gross pathology

Fetal body weight changes:

effects observed, treatment-related

Description (incidence and severity):

Mean fetal weights were similar in the fetuses from the 20 mg/kg bw/day-treated and control dams.

Test-item related adverse effects were noted on the mean fetal weights at 100 and 250 mg/kg bw/day, with a dose related pattern and mean values of 13% and 32% lower than controls, respectively (p<0.01). The effects on fetal body weight were attributed to intrauterine growth retardation.

Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): not examined

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

Most fetuses examined were viable and no effects attributable to Tea Tree Oil treatment were noted in the mean number of viable fetuses/group.

Changes in sex ratio:

no effects observed

Description (incidence and severity):

No effects attributable Tea Tree Oil treatment were noted in the sex distribution. The sex ratios were similar in the control and treated groups when evaluated per litter.

Changes in litter size and weights:

not examined

Changes in postnatal survival:

not examined

External malformations:

effects observed, treatment-related

Description (incidence and severity):

Malformations such as generalized edema (3 litters) or short maxilla (1 fetus, 1 litter) were noted in the 250 mg/kg bw/day dose group. No macroscopic findings were noted in the appearance of the placenta with the exception of one female that had placental fibrinoid degeneration, associated with 2 malformed fetuses.

Skeletal malformations:

effects observed, treatment-related

Description (incidence and severity):

A statistically higher incidence of skeletal malformations (short, bent scapula or humerus, short and bent femur, malformed vertebrae) unrelated to intrauterine growth retardation occurred only at 250 mg/kg bw. There were 7 affected fetuses (9%) at 250 mg/kg bw, compared to 0 in the control group (p<0.01).

Visceral malformations:

no effects observed

Description (incidence and severity):

There was no statistically significant difference from control in the number of visceral malformations. A finding of hypoplastic thymus in 1 mid dose and 2 high dose fetuses was not attributable to Tea Tree Oil treatment.

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Skeletal variations: A statistically higher number of skeletal variations were noted at 100 and 250 mg/kg bw/day. When evaluated on a per group basis, the total number of fetuses with variations were 44 and 62 in the mid and high dose groups, respectively, versus 16 in the control group (41% and 78% of the fetuses evaluated, versus 14% in the control group, p<0.01). The increased incidence of skeletal variations in the 100 and 250 mg/kg dose groups were concluded to be secondary to maternal toxicity, including intrauterine growth retardation.

Visceral variations: Visceral variations were statistically higher at 250 mg/kg bw with 15 affected foetuses in the high dose group, versus 4 in the control group (19% of the fetuses evaluated, versus 3% in the control group, p<0.01). Visceral variations including dilated brain ventricles and displaced gonads associated with the intrauterine growth retardation were noted. In addition, variations such as small nasal conchae, close origin of brachiocephalic and carotid, dilated ureter or dilated renal pelvis were statistically increased at 250 mg/kg bw/day.

Details on embryotoxic / teratogenic effects:

No adverse effects related to Tea Tree Oil Treatment were observed in the 20 mg/kg bw/day treatment group. In all cases, adverse effects seen in the 100 and 250 mg/kg bw/day groups are considered to be secondary to severe maternal toxicity and cannot be regarded as evidence of embrotoxicity/teratogenicity.

Dose descriptor:

NOAEL

Effect level:

20 mg/kg bw/day

Based on:

test mat.

Sex:

not specified

Basis for effect level:

fetal/pup body weight changes

Remarks on result:

other:

Remarks:

Effects at 100 and 250 mg/kg bw/day secondary to maternal toxicity

Dose descriptor:

NOAEL

Effect level:

100 mg/kg bw/day

Based on:

test mat.

Sex:

not specified

Basis for effect level:

external malformations

Remarks on result:

other:

Remarks:

Effects at 250 mg/kg bw/day secondary to maternal toxicity

Dose descriptor:

NOAEL

Effect level:

100 mg/kg bw/day

Based on:

test mat.

Sex:

not specified

Basis for effect level:

skeletal malformations

Remarks on result:

other:

Remarks:

Effects at 250 mg/kg bw/day secondary to maternal toxicity.

Abnormalities:

effects observed, treatment-related

Localisation:

skeletal: vertebra

Description (incidence and severity):

Malformed vertebrae in 2/80 fetuses examined (3%)

Abnormalities:

effects observed, treatment-related

Localisation:

other: Skeletal: Scapula and/or humerus

Description (incidence and severity):

Short, bent scapula and/or humerus in 4/80 fetuses examined (5%)

Abnormalities:

effects observed, treatment-related

Localisation:

skeletal: hindlimb

Description (incidence and severity):

Short, bent femur in 2/80 fetuses examined (3%)

Abnormalities:

effects observed, treatment-related

Localisation:

other: Generalised edema

Description (incidence and severity):

Generalised oeedema seen in three litters

Abnormalities:

effects observed, treatment-related

Localisation:

external: face

Description (incidence and severity):

Short maxilla (1 fetus, 1 litter)

Developmental effects observed:

yes

Lowest effective dose / conc.:

100 mg/kg bw/day

Treatment related:

yes

Relation to maternal toxicity:

developmental effects as a secondary non-specific consequence of maternal toxicity effects

Dose Formulation Analysis Results:

All formulations proved to be homogeneous, as similar results were obtained from samples taken from the top, middle and bottom of the formulations, and the measured concentrations were found to be in the acceptable range of 100±10% (94 to 108%) of the nominal concentrations. No peak was detected in the control samples at the retention time corresponding to that of (+/-) terpinen-4-ol.

Conclusions:

Tea tree oil administered to naturally mated, assumed-pregnant Hannover Wistar female rats at dose levels of 20, 100 and 250 mg Tea Tree Oil/kg bw/day, from gestation days 5 to 19, resulted in mortality at 250 mgl/kg bw/day. Severe maternal toxicity was seen in dams from the 100 and 250 mg/kg bw/day groups, as evidenced by clinical signs, reduced food consumption and weight gain reductions of 20% and 45%, respectively, over the gestation period. Fetal abnormalities seen in the 100 and 250 mg/kg bw/day groups were considered to be secondary to maternal toxicity.
The NOAEL for Tea Tree Oil for developmental toxicity (secondary to severe maternal toxicity) was 20 mg/kg bw/day. The NOAEL for maternal toxicity was 20 mg/kg bw/day. This value for maternal toxicity is carried forward for development of the DNEL.

Executive summary:

A GLP compliant developmental toxicity study was conducted with Tea Tree Oil (TTO) in naturally mated, assumed pregnant Hannover Wistar female rats according to OECD Test Guideline 414, to evaluate the effect on dams and developing conceptuses after oral (gavage) administration during pregnancy. A control group which received PEG 400 only and three groups treated with TTO formulated in PEG 400 at 250, 100 and 20 mg/kg bw/day were included in the study. TTO formulated in PEG 400 was administered daily from gestation day (GD) 5 to GD19, where GD0 was considered the day of mating. Caesarean section and maternal necropsy with macroscopic examination were performed on GD20 in all the females surviving to termination. Placentas and fetuses were examined macroscopically and fetal body weight was measured. The gender of each fetus was determined. Thereafter, approximately half of each litter was subjected to a visceral examination and the remaining fetuses were processed for skeletal examination.

At 250 mg TTO/kg bw/day, mortality occurred in 7/27 females between GD8 and GD11. Clinical signs in animals that died included noisy respiration, decreased activity and/or piloerection. Clinical signs in surviving animals included decreased activity, hunched back position, noisy respiration, piloerection, red spots on the tail and/or soft feces. Treatment at 100 mg/kg bw/day resulted in no mortality but clinical signs such as noisy respiration, decreased activity, hunched back position, red spots and/or soft feces were noted in 13/26 females. At 20 mg TTO/kg bw/day, there was no mortality. Noisy respiration or soft feces were occasionally noted, but were considered not to be toxicologically relevant.

Severely reduced maternal body weight gain (-20% and -45% respectively, when compared to control) and food consumption were noted at 100 and 250 mg TTO/kg bw/day.  

In females treated at 250 mg TTO/kg bw/day, bilateral enlarged adrenals were observed in all animals found dead and in 6/20 of animals that survived until scheduled necropsy. This finding was attributed to treatment. A single animal in the mid dose group also had bilateral enlarged adrenals.

At 250 mg TTO/kg bw/day, there was a higher number of late embryonic deaths and consequently post-implantation loss, leading to an overall higher total intrauterine mortality. Post-implantation losses were unchanged in the low and mid treatment groups. Post-implantation mortality was considered secondary to maternal toxicity.

Statistically lower mean gravid uterine weight was noted at 250 mg TTO/kg bw/day and lower terminal mean body weights when corrected for the gravid uterine weight, were noted at 100 and 250 mg TTO/kg bw/day. The corrected mean body weight gains were lower than the controls in the two highest dose groups. These adverse effects were considered to be related to TTO administration.

Most fetuses were viable and no effects related to TTO were noted in the mean number of viable fetuses/group, or their sex distribution. The sex ratios were similar in the control and treated groups when evaluated per litter.

Adverse effects were noted in mean fetal weights at 100 and 250 mg TTO/kg bw/day, with a dose-related pattern. The effects on fetal body weight were related to intrauterine growth retardation. External abnormalities such as local edema in the cervical area, generalized edema or short maxilla were noted in the high dose group.

There was no statistically significant difference from control in the number of visceral malformations. A statistically higher number of visceral variations were noted in the 250 mg/kg bw/day dose group. These included dilated brain ventricles and displaced gonads associated with the intrauterine growth retardation were noted. In addition, variations such as small nasal conchae, close origin of brachiocephalic and carotid, dilated ureter or dilated renal pelvis were statistically increased at 250 mg/kg bw/day.

A statistically higher incidence of skeletal malformations unrelated to intrauterine growth retardation was noted in the 250 mg/kg bw/day group. This included displaced rib cartilages at the sternum, malformed vertebrae, and/or short, bent scapula, humerus or femur. Statistically higher numbers of skeletal variations, secondary to maternal toxicity, were noted at 100 and 250 mg/kg bw/day.

In summary, severe maternal toxicity was seen in dams from the 100 and 250 mg/kg bw/day groups, as evidenced primarily by clinical signs, reduced food consumption and reduced weight loss gains (and mortality at the high dose). Fetal abnormalities seen in the 100 and 250 mg/kg bw/day groups were secondary to maternal toxicity. The NOAEL for Tea Tree Oil for developmental toxicity (secondary to severe maternal toxicity) was 20 mg/kg bw/day. The NOAEL for maternal toxicity was 20 mg/kg bw/day.  This value for maternal toxicity is carried forward for development of the DNEL.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

20 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

This study has been assigned a reliability of 1 and meets the information requirements of REACH.

Justification for classification or non-classification

A 28-day developmental toxicity study was conducted in Hannover Wistar rats (Kubaszky R 2011).  Adverse effects, seen only in developing foetuses at doses of 100 and 250 mg/kg bw/day, were secondary to severe maternal toxicity at these dose levels, as evidenced primarily by clinical signs, reduced food consumption and reduced weight loss gains in the dams (with mortality at the high dose).

The NOAEL for tea tree oil for developmental toxicity was 20 mg/kg bw/day.  As effects seen at 100 and 250 mg/kg bw/day were a secondary non-specific consequence of severe maternal toxicity, it is concluded that tea tree oil does not meet the criteria for classification as a reproductive toxicant.

The NOAEL for maternal toxicity was 20 mg/kg bw/day.  As no significant, organ-specific adverse effects were observed at the CLP guidance values required for classification as STOT RE, it is concluded that tea tree oil does not meet the criteria for classification on the basis of repeat dose toxicity.  This conclusion is supported by the results of the available 28-day oral repeat dose test (see IUCLID section 7.5).  The repeat-dose NOAEL identified for maternal toxicity is carried forward for development of the DNEL.