Amides, C12-18 and C18-unsatd., N,N-bis(hydroxyethyl)

Endpoint summary

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Effects on fertility

Link to relevant study records

Reference

Reference 1

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

read-across based on grouping of substances (category approach)

Adequacy of study:

key study

Study period:

31 January 2013 - 17 June 2013

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

KL2 due to RA

Justification for type of information:

Refer to section 13 of IUCLID for details on the category justification.

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: breeder: Charles River Laboratories Italia, Calco, Italy
- Age/weight at study initiation: on the first day of treatment, the males were approximately 10 weeks old and had a mean body weight of 388 g (range: 335 g to 438 g) and the females were approximately 9 weeks old had a mean body weight of 236 g (range: 203 g to 270 g)
- Housing: the animals were individually housed, except during pairing and lactation, in polycarbonate cages
- Diet: SSNIFF R/M-H pelleted diet (free access)
- Water: tap water filtered with a 0.22 µm filter (free access)
- Acclimation period: 8 days before the beginning of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22±2°C
- Humidity (%): 50±20%
- Air changes (per hr): approximately 12 cycles/hour of filtered, non-recycled air
- Photoperiod (hrs dark / hrs light): 12 h/12 h.

IN-LIFE DATES: 13 February 2013 to 09 April 2013

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test item was administered as a suspension in the vehicle. The dose formulations were prepared according to the following process, as described in an homogeneity/stability study:
- a small amount of test item was melt using water bath at a temperature of +50°C,
- the vehicle was warmed at +50°C using water bath,
- the appropriate amount of melt test item was weighed in the preparation beaker and the corresponding amount of vehicle was added,
- the test item and the vehicle were mixed using magnetic stirrer in the water bath at +50°C during 10 minutes,
- the obtained suspension was stirred at ambient temperature at least 30 minutes.

No correction factor was applied.
The test item dose formulations were prepared on a weekly basis, stored at room temperature protected from light prior to use and delivered to the study room at room temperature and protected from light.

When not on the day of formulation preparation, test item formulations were warmed under magnetic stirring at +50°C using water bath during at least 30 minutes and then kept under magnetically stirring at ambient temperature for at least 30 minutes before daily delivery to the study room.

VEHICLE
- Choice of vehicle: good suspension in corn oil
- Concentration in vehicle: 20, 60 and 200 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg/day.

Details on mating procedure:

- M/F ratio per cage: 1/1
- Length of cohabitation (mating period): until mating occurred
- Proof of pregnancy: vaginal plug or sperm in the morning vaginal lavage referred as day 0 post-coitum
- After successful mating each pregnant female was caged individually

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Type of method: GC-FID
Test item concentrations: remained within an acceptable range of variation compared to nominal values.
Homogeneity: assessed in homogeneity study (satisfactory results)
Stability: assessed in stability study (stable after 9 days at room temperature and protected from light)

Duration of treatment / exposure:

In the males:
− 2 weeks before mating,
− during the mating period,
− until sacrifice (at least 5 weeks in total),

In the females:
− 2 weeks before mating,
− during the mating period (1 week),
− during pregnancy,
− during lactation until day 5 post-partum inclusive,
− until sacrifice for females which had not delivered.

Frequency of treatment:

Daily

Details on study schedule:

- No F1 parents (only one generation mated)
- Age at mating of the mated animals in the study: 11-12 weeks

Dose / conc.:

0 mg/kg bw/day (actual dose received)

Remarks:

Controls

Dose / conc.:

100 mg/kg bw/day (actual dose received)

Dose / conc.:

300 mg/kg bw/day (actual dose received)

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

10 animals per sex per dose.

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose-levels were selected in agreement with the Sponsor, following the results of a previous 2-week toxicity study. In this study, three groups of three male and three female Sprague-Dawley rats received the test item as a suspension in corn oil at 100, 300 or 1000 mg/kg/day bw for 2 weeks by gavage. There were no unscheduled deaths. Reduced mean body weight gain and mean food consumption were noted in males during the first week of treatment. Clinical signs were ptyalism in all test item-treated animals and hunched posture in two males and one female at the high-dose during the second week of treatment. There were no test item-related macroscopic findings.

- Animal assignment: computerized stratification procedure.

Positive control:

no (not required).

Parental animals: Observations and examinations:

MORTALITY/MORBIDITY:
- Time schedule: at least twice a day during the treatment period.

CLINICAL OBSERVATIONS:
- Time schedule: once a day during the treatment period.

DETAILED CLINICAL SIGNS:
- Time schedule: once a week during the treatment period.

BODY WEIGHT:
- Time schedule: Males: on the first day of treatment, then once a week until sacrifice. Females: on the first day of treatment, then once a week until mating (or until sacrifice), on Days 0, 7, 14 and 20 post-coitum and days 1 and 5 post-partum.

FOOD CONSUMPTION:
- Time schedule: Males: once a week until sacrifice. Females: once a week until mating, on Days 0, 7, 14 and 20 post-coitum and days 1 and 5 post-partum.

- NEUROBEHAVIOURAL EXAMINATION:
- Time schedule: at the end of the treatment period.

HAEMATOLOGY:
- Time schedule: on the day of sacrifice.

CLINICAL CHEMISTRY:
- Time schedule: on the day of sacrifice.

REPRODUCTION (apart from indices):
- Pre-coital time and duration of gestation were recorded.

Oestrous cyclicity (parental animals):

Fresh vaginal lavage (stained with methylene blue), each morning during the pairing period, until females are mated.

Sperm parameters (parental animals):

Parameters examined in males of parental generation:
- weighing and microscopic examination:yes
- special emphasis to the spermatogenic cycle and testicular interstitial cells (groups 1 and 4).

Litter observations:

STANDARDISATION OF LITTERS: No

PARAMETERS EXAMINED:
- number and sex of pups,
- number of live, dead and cannibalized pups,
- presence of gross anomalies, weight gain, clinical signs.

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: all surviving animals after the end of the mating period
- Female animals: all surviving animals = day 6 post-partum or, for females which had not delivered yet, day 25 post-coitum.

ORGAN WEIGHTS: Yes

GROSS PATHOLOGY:
Complete macroscopic post-mortem examination.

HISTOPATHOLOGY:
- on all tissues listed in the table below for the first five control and high dose animals (groups 1 and 4) sacrificed as scheduled,
- on all macroscopic lesions,
- all females sacrificed because of no delivery to investigate possible causes,
- liver (both sexes), thymus (females only), seminal vesicles (males only) and bone marrow (females only) from the first five sacrificed as scheduled males and the first five females sacrificed on day 6 p.p. of the low- and intermediate-dose groups (groups 2 and 3).

Postmortem examinations (offspring):

SACRIFICE: on day 5 post-partum

GROSS NECROPSY: on all pups (surviving and found dead)

HISTOPATHOLOGY: No

ORGAN WEIGTHS: No

Reproductive indices:

Pre-implantation loss = 100 * (Number of corpora lutea - Number of implantation sites) / Number of corpora lutea
Post-implantation loss = 100 * (Number of implantation sites - Number of live pups) / Number of implantations
Mating index = 100 * (Number of mated animals / Number of paired animals)
Fertility index = 100 * (Number of pregnant female partners / Number of mated pairs)
Gestation index = 100 * (Number of females with live born pups / Number of pregnant females)

Offspring viability indices:

Live birth index = 100 * (Number of live born pups / Number of delivered pups)
Viability index on day 4 p.p. = 100 * (Number of surviving pups on day 4 p.p. / Number of live born pups)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Ptyalism, considered of minor toxicological significance, was observed in all animals at 300 and 1000 mg/kg/day bw from the first or second week of treatment and in most of the animals at 100 mg/kg/day bw but later. Hypoactivity, loud breathing, piloerection and/or round back observed for some days in a few animals at 300 mg/kg/day bw but more at 1000 mg/kg/day bw were considered to be of limited toxicological significance. Incidental findings consisted of half-closed eyes, reflux at dosing, cutaneous lesion and abnormal growth of teeth.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

There were no deaths considered as test item-related. One female given 1000 mg/kg/day bw was prematurely sacrificed on day 23 p.c. due to difficulty to deliver. Piloerection, round back, cold to the touch, abdominal breathing, reddish vaginal discharge, chromodacryorrhea and chromorhinorrhea were observed on day 22 and/or 23 p.c. Blood in the bedding, fetuses (mostly dead) and a few placentae were found in the bedding on day 23 p.c. This death was considered to be incidental. There were no other unscheduled deaths during the study.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

There were no relevant effects on mean body weights and mean body weight gains. The low mean body weight gain in high-dose males for the interval days 1 to 8 when compared to controls was considered to be of no toxicological significance (no statistical level and transient decrease).

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

In males, mean food consumption at 1000 mg/kg/day bw was statistically significantly reduced in the first week of treatment when compared with controls (which correlated with a non-statistically significantly lower mean body weight gain at that time). It became similar to controls in the second week of dosing. This slight and transient effect was considered to be of limited toxicological significance. Mean food consumption at 100 and 300 mg/kg/day bw was not affected. Mean food consumption in females was considered to be unaffected by the test item treatment.

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

APPT values were not considered to be affected (one control data in males was higher than the others hence the shortened mean values in the test item-treated groups; there was no dose-relationship in females). For the slightly higher mean white blood cell counts when compared with controls, a relationship with the test item treatment was considered to be doubtful (no clear dose-relationship, individual values generally included in historical control data and no statistical level).

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Mean cholesterol level was higher in test item-treated female groups in a dose-related manner, reaching statistical and toxicological significance at 300 and 1000 mg/kg/day bw. All individual values in both groups were included in historical control data and in absence of adverse correlates in the study, this was considered to be non-adverse. There was no dose relationship in males, but an effect of the test item may be suspected in two males treated at 1000 mg/kg/day bw which had a high cholesterol level (2.3 and 2.4 mmol/L). Mean sodium concentration was also statistically significant higher in females treated at 1000 mg/kg/day bw when compared with controls. Males or other electrolytes in females were not affected and the variation was not severe. Therefore, this finding was not considered to be of toxicological significance.An effect of the test item treatment on mean albumin concentration at 300 mg/kg/day bw in both sexes was considered unlikely as there was no dose-relationship. The increase observed in triglyceride levels at 300 and 1000 mg/kg/day bw in both sexes was considered to be incidental as there was no statistical level reached for the group means and no dose-relationship seen in individual data. Moreover, the increase at 1000 mg/kg/day bw was due to isolated animals per sex only.

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

There were no relevant findings on FOB in test item-treated groups when compared to the control group. An effect of the test item treatment in males and females at 300 and/or 1000 mg/kg/day bw on mean motor activity data was considered to be equivocal but of limited toxicological significance. The vast majority of the individual data were similar to what can usually be seen in this type of study.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Test item-related changes were observed in the liver and the thymus.

Liver
Minimal hepatocellular hypertrophy was seen in 1/5 males given the test item at 300 mg/kg/day bw and 4/6 males and 2/5 females given 1000 mg/kg/day bw. This non-adverse change was considered to be test item-related and correlated with the higher weight noted at necropsy. Other changes seen in the liver, including the minimal foci of subcapsular necrosis seen in 1/5 control females, 1/5 males at 300 mg/kg/day bw and 2/5 females at 1000 mg/kg/day bw were considered to be fortuitous and without any relationship with the test item.

Thymus
Minimal or slight lymphoid atrophy was seen in 2/5 females given 1000 mg/kg/day bw, correlating with the lower weight at necropsy. Any relationship with the test item was considered to be likely but non-adverse (low number of animals affected and low grades). No test item related changes were seen at 100 or 300 mg/kg/day bw.

Miscellaneous changes
- Seminal vesicles
Minimal apoptosis was seen in 1/5 males given 100 mg/kg/day bw and 3/6 animals given 1000 mg/kg/day bw. As this change was minimal, and in the absence of a dose-related trend and other test item-related changes in testes or epididymides, any relationship with the test item was considered to be unlikely.

- Bone marrow
There was a trend towards higher presence of adipose tissue in the bone marrow of females at all dose levels when compared with controls. As this was not seen in males, poorly dose-related and as this was not correlated with any changes in the hematological parameters, any relationship with the test item was excluded.

- Lungs
Mucus was seen in bronchi and/or bronchioli of 3/6 males given 1000 mg/kg/day bw and eosinophilic infiltrate in 3/6 males at this dose-level. Increased alveolar macrophages were seen at a similar incidence and severity between controls and treated animals in both sexes. These changes were consistent with aspiration or regurgitation of the oily vehicle or test item during the technical gavage procedures. Any direct relationship with the test item was excluded.

A careful examination of testes, epididymides, and female genital tract did not show any test item-related effects on these organs. Other histopathological changes were considered to be part of the normal background of changes commonly seen in rats and without any relationship with the test item.

Reproductive performance:

no effects observed

Description (incidence and severity):

Mating and fertility data
There were no test item-related effects on mean mating and fertility data. At 1000 mg/kg/day bw, one female was blocked in metestrous/diestrous for 12 days and mated with its male after 13 days, and one female was prematurely sacrificed on day 23 p.c. because of difficulty to deliver as previously mentioned. Both events were considered to be incidental as isolated. A total of five females (three controls and two females treated at 1000 mg/kg/day bw) were sacrificed on day 25 p.c. because of no delivery. They were non-pregnant.

Delivery data
There were no test item-related effects on mean delivery data. Mean pre- and post-implantation loss data were lower than the highest percentages seen in historical control data, thus there were no effects of the test item treatment.

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Parental toxicity (non adverse)

Key result

Dose descriptor:

NOEL

Effect level:

ca. 1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Reproductive performance (mating and fertility)

Key result

Critical effects observed:

not specified

Clinical signs:

no effects observed

Description (incidence and severity):

None of the clinical signs were considered to be test item related (comparable incidences in control pups).

Mortality / viability:

no mortality observed

Description (incidence and severity):

There were no effects on pup viability.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There were no effects of the test item treatment on mean body weight and mean body weight changes in pups. The trend toward higher mean body weights and mean body weight gain at 100 mg/kg/day bw was considered to be incidental (not dose-related).

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Both findings are not very often recorded in controls of such studies, although they can appear spontaneously. An effect of the test item treatment was suspected since they were observed with a dose relationship at both the highest dose-levels. However, the effect was considered as of minor toxicological significance as these findings are generally considered as morphological variations (not malformations) and as their incidence was not severe.

Other effects:

no effects observed

Description (incidence and severity):

SEX RATIO
Despite the fact that the pup sex ratio at 1000 mg/kg/day bw was slightly above the historical control range, it was considered to be of no toxicological significance in view of its low amplitude.

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Toxic effects on progeny (minor toxicological significance)

Key result

Critical effects observed:

not specified

Key result

Reproductive effects observed:

no

Conclusions:

Under the study conditions, the NOAEL for parental toxicity, the NOEL for reproductive performance (mating and fertility) and the NOAEL for toxic effects on progeny were considered to be 1000 mg/kg/day bw.

Executive summary:

A study was conducted to evaluate the repeated dose oral toxicity of the read across substance, C18-unsatd. MIPA, according to OECD Guideline 422, in compliance with GLP. Groups of ten male and ten female Sprague-Dawley rats received the read across substance at dose-levels of 0, 100, 300 or 1000 mg/kg bw/day daily by oral (gavage) administration 2 weeks before mating, during mating, gestation and until up to Day 5 p.p. The concentration of the dose formulation was checked in study Weeks 1, 3 and 6. The animals were checked at least twice daily during the dosing period for mortality and morbidity and once daily for clinical signs. Detailed clinical observations were performed once a week. Body weight and food consumption were recorded once a week during premating and mating periods (food consumption not during mating), and during gestation on Days 0, 7, 14 and 20 p.c. and lactation on Days 1 and 5 p.p. The animals were paired for mating after 2 weeks of treatment and the females were allowed to litter and rear their progeny until Day 5 p.p. The total litter sizes and the sex of each pup were recorded after birth. The pups were observed daily for clinical signs, abnormal behaviour and external abnormalities and weighed on Days 1 and 5 p.p. At the end of the treatment period, Functional Observation Battery, motor activity and laboratory investigations (hematology and blood biochemistry) were carried out on five males and five females. The males were sacrificed after at least 5 weeks of treatment and the females on Day 6 p.p. Final body weights and selected organs weights (adrenals, brain, epididymides, heart, kidneys, liver, spleen, testes and thymus) were recorded and a complete macroscopic post-mortem examination was performed, with particular attention paid to the reproductive organs. A microscopic examination was performed on selected organs from five males and five females in the control- and high-dose groups, on liver, thymus, seminal vesicles and bone marrow from five males and/or five females in the low- and intermediate-dose groups and on all macroscopic lesions. The pups were sacrificed on Day 5 p.p. and submitted for a macroscopic post-mortem examination of the principal thoracic and abdominal organs. The read across substance concentrations checked during the study were within an acceptable range of variations when compared to the nominal values (± 15%). There was no read across substance in control formulations. There were no read across substance-related deaths. Clinical signs consisted of ptyalism in all animals at 300 and 1000 mg/kg bw/day bw and in most of the animals at 100 mg/kg bw/day bw (minor toxicological significance), as well as hypoactivity, loud breathing, piloerection and/or round back observed in a few animals at 300 and 1000 mg/kg bw/day bw for a few days (limited toxicological significance). There were no relevant effects on mean body weight, mean Functional Observation Battery (FOB), as well as on mean hematology parameters in any group and sex. An effect of the read across substance treatment on mean motor activity data at 300 and/or 1000 mg/kg bw/day bw was considered to be equivocal but of limited toxicological significance. In males, mean food consumption at 1000 mg/kg bw/day bw was reduced in the first week of treatment only (23 g/male/day, vs. 27, p<0.001). This effect was considered to be of limited toxicological significance. Mean food consumption in males at 100 and 300 mg/kg bw/day bw and in females were not affected. In females, mean cholesterol level was higher than in controls at 300 and 1000 mg/kg bw/day bw (up to 1.9 mmol/L, vs.1.2, p<0.01) and considered to be non-adverse in absence of adverse correlates in the study. There were no relevant blood biochemistry findings in females at 100 mg/kg bw/day bw or in males. At histopathology at 1000 mg/kg bw/day bw, minimal hepatocellular hypertrophy correlating with higher mean liver weight was seen in the liver of both sexes (about +28% in males and +20% in females compared to controls, p<0.01 generally). In females, mild lymphoid atrophy was seen in the thymus of 2/5 females, correlating with lower mean weight at necropsy (about -22% from controls). At 300 mg/kg bw/day bw, only minimal hepatocellular hypertrophy was seen in the liver of a single male correlating with minor higher mean absolute weight in this group. All these microscopic findings were considered to be non-adverse (low number of animals affected and/or minimal to slight grades). There were no histopathological effects at 100 mg/kg bw/day bw. Under the study conditions, the NOAEL for parent systemic toxicity was considered to be 1000 mg/kg bw/day (Bentz, 2013).

Reference 2

Endpoint:

extended one-generation reproductive toxicity - basic test design (Cohorts 1A, and 1B without extension)

Type of information:

read-across based on grouping of substances (category approach)

Adequacy of study:

key study

Study period:

As of 2022 at the earliest. Depending on the results of the other planned studies (e.g. OECD TG 421, 408, 414) and testing proposal acceptance.

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

KL2 due to RA

Justification for type of information:

TESTING PROPOSAL ON VERTEBRATE ANIMALS

NON-CONFIDENTIAL NAME OF SUBSTANCE:
- Amides, C8-18 (even numbered) and C18-unsatd., N,N-bis(hydroxyethyl (C8-18 and C18-unsatd. DEA), EC No. 931-329-6

CONSIDERATIONS THAT THE GENERAL ADAPTATION POSSIBILITIES OF ANNEX XI OF THE REACH REGULATION ARE NOT ADEQUATE TO GENERATE THE NECESSARY INFORMATION [please address all points below]:
- Available GLP studies: none
- Available non-GLP studies: none
- Historical human data: none
- (Q)SAR: not applicable
- In vitro methods: not applicable
- Weight of evidence: not sufficient
- Grouping and read-across: not sufficient
- Substance-tailored exposure driven testing [if applicable]: none
- Approaches in addition to above [if applicable] : not applicable
- Other reasons [if applicable]: none

CONSIDERATIONS THAT THE SPECIFIC ADAPTATION POSSIBILITIES OF ANNEXES VI TO X (AND COLUMN 2 THEREOF) OF THE REACH REGULATION ARE NOT ADEQUATE TO GENERATE THE NECESSARY INFORMATION:
- Proposed adaptations were not considered sufficient to address this endpoint. This was discussed with ECHA in the frame of a Dossier Improvement Action Plan (DIAP).

FURTHER INFORMATION ON TESTING PROPOSAL IN ADDITION TO INFORMATION PROVIDED IN THE MATERIALS AND METHODS SECTION:
- Proposed study design: according to OECD Guideline 443

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes

Justification for study design:

SPECIFICATION OF STUDY DESIGN FOR EXTENDED ONE-GENERATION REPRODUCTION TOXICITY STUDY WITH JUSTIFICATIONS

- Premating exposure duration for parental (P0) animals : to be decided
- Basis for dose level selection : will be based on the outcome of planned repeated dose toxicity studies
- Inclusion/exclusion of extension of Cohort 1B : will be based on the outcome of planned repeated dose toxicity studies
- Termination time for F2 : will be based on the outcome of planned repeated dose toxicity studies
- Inclusion/exclusion of developmental neurotoxicity Cohorts 2A and 2B : will be based on the outcome of planned repeated dose toxicity studies
- Inclusion/exclusion of developmental immunotoxicity Cohort 3 : will be based on the outcome of planned repeated dose toxicity studies
- Route of administration : oral

Species:

rat

Sex:

male/female

Route of administration:

oral: gavage

Dose descriptor:

NOAEL

Remarks on result:

other: Testing planned

Critical effects observed:

not specified

Dose descriptor:

NOAEL

Remarks on result:

other: Testing planned

Critical effects observed:

not specified

Reproductive effects observed:

not specified

Reference 3

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

read-across based on grouping of substances (category approach)

Adequacy of study:

supporting study

Study period:

2020-Ongoing

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

KL2 due to RA

Justification for type of information:

Refer to section 13 of IUCLID for details on the category justification.

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

GLP compliance:

yes (incl. QA statement)

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Remarks:

0.5% aqueous solution of carboxymethylcellulose

Analytical verification of doses or concentrations:

yes

Dose / conc.:

0 mg/kg bw/day (nominal)

Dose / conc.:

250 mg/kg bw/day (nominal)

Dose / conc.:

500 mg/kg bw/day (nominal)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

10 male and 10 female

Control animals:

yes, concurrent vehicle

Dose descriptor:

NOAEL

Remarks on result:

other: Testing ongoing

Critical effects observed:

not specified

Dose descriptor:

NOAEL

Remarks on result:

other: Testing ongoing

Critical effects observed:

not specified

Reproductive effects observed:

not specified

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

A study was conducted to evaluate the repeated dose oral toxicity of the read across substance, C18-unsatd. MIPA, according to OECD Guideline 422, in compliance with GLP. Groups of ten male and ten female Sprague-Dawley rats received the read across substance at dose-levels of 0, 100, 300 or 1000 mg/kg bw/day daily by oral (gavage) administration 2 weeks before mating, during mating, gestation and until up to Day 5 p.p. The concentration of the dose formulation was checked in study Weeks 1, 3 and 6. The animals were checked at least twice daily during the dosing period for mortality and morbidity and once daily for clinical signs. Detailed clinical observations were performed once a week. Body weight and food consumption were recorded once a week during premating and mating periods (food consumption not during mating), and during gestation on Days 0, 7, 14 and 20 p.c. and lactation on Days 1 and 5 p.p. The animals were paired for mating after 2 weeks of treatment and the females were allowed to litter and rear their progeny until Day 5 p.p. The total litter sizes and the sex of each pup were recorded after birth. The pups were observed daily for clinical signs, abnormal behaviour and external abnormalities and weighed on Days 1 and 5 p.p. At the end of the treatment period, Functional Observation Battery, motor activity and laboratory investigations (hematology and blood biochemistry) were carried out on five males and five females. The males were sacrificed after at least 5 weeks of treatment and the females on Day 6 p.p. Final body weights and selected organs weights (adrenals, brain, epididymides, heart, kidneys, liver, spleen, testes and thymus) were recorded and a complete macroscopic post-mortem examination was performed, with particular attention paid to the reproductive organs. A microscopic examination was performed on selected organs from five males and five females in the control- and high-dose groups, on liver, thymus, seminal vesicles and bone marrow from five males and/or five females in the low- and intermediate-dose groups and on all macroscopic lesions. The pups were sacrificed on Day 5 p.p. and submitted for a macroscopic post-mortem examination of the principal thoracic and abdominal organs. The read across substance concentrations checked during the study were within an acceptable range of variations when compared to the nominal values (± 15%). There was no read across substance in control formulations. There were no read across substance-related deaths. Clinical signs consisted of ptyalism in all animals at 300 and 1000 mg/kg bw/day bw and in most of the animals at 100 mg/kg bw/day bw (minor toxicological significance), as well as hypoactivity, loud breathing, piloerection and/or round back observed in a few animals at 300 and 1000 mg/kg bw/day bw for a few days (limited toxicological significance). There were no relevant effects on mean body weight, mean Functional Observation Battery (FOB), as well as on mean hematology parameters in any group and sex. An effect of the read across substance treatment on mean motor activity data at 300 and/or 1000 mg/kg bw/day bw was considered to be equivocal but of limited toxicological significance. In males, mean food consumption at 1000 mg/kg bw/day bw was reduced in the first week of treatment only (23 g/male/day, vs. 27, p<0.001). This effect was considered to be of limited toxicological significance. Mean food consumption in males at 100 and 300 mg/kg bw/day bw and in females were not affected. In females, mean cholesterol level was higher than in controls at 300 and 1000 mg/kg bw/day bw (up to 1.9 mmol/L, vs.1.2, p<0.01) and considered to be non-adverse in absence of adverse correlates in the study. There were no relevant blood biochemistry findings in females at 100 mg/kg bw/day bw or in males. At histopathology at 1000 mg/kg bw/day bw, minimal hepatocellular hypertrophy correlating with higher mean liver weight was seen in the liver of both sexes (about +28% in males and +20% in females compared to controls, p<0.01 generally). In females, mild lymphoid atrophy was seen in the thymus of 2/5 females, correlating with lower mean weight at necropsy (about -22% from controls). At 300 mg/kg bw/day bw, only minimal hepatocellular hypertrophy was seen in the liver of a single male correlating with minor higher mean absolute weight in this group. All these microscopic findings were considered to be non-adverse (low number of animals affected and/or minimal to slight grades). There were no histopathological effects at 100 mg/kg bw/day bw. Under the study conditions, the NOAEL for parent systemic toxicity was considered to be 1000 mg/kg bw/day (Bentz, 2013).

Also, a reproductive and developmental screening study according to OECD Guideline 421 is currently ongoing with the read across substance C8-18 and C18-unsatd. DEA in order to further support the read across approach proposed for the FAA category members.

In addition, after discussion with ECHA in the frame of a Dossier Improvement Action Plan (DIAP), a testing proposal is submitted for the conduct of an extended one generation reproductive toxicity study (EOGRTS) according to OECD Guideline 443 with C8-18 and C18-unsatd. DEA.

Effects on developmental toxicity

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Official Journal of European Community L 133, May 30, 1988; 87/302/EEC

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Official Journal of European Community L 180, March 01, 1991; 91/325/EEC

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River, Wiga D-Sulzfeld
- Age at study initiation: 8-10 wk
- Weight at study initiation: 209 g (mean)
- Housing: Single animal in Makrolon Type M3 cage (Ebeco) with standard softwood bedding
- Diet: Pelleted Altromin Maintenance Diet 1324, ad libitum
- Water: Tap water, ad libitum
- Acclimation period: 5 d

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 43-66
- Air changes (per h): 10-15
- Photoperiod (h dark/h light): 12/12

Route of administration:

oral: gavage

Vehicle:

arachis oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Test material was suspended in Arachidis oil, DAB 9 such that the required dose per kg body weight was contained in 5 mL.

VEHICLE
Concentration in vehicle: 0, 20, 60 and 200 mg/mL

Analytical verification of doses or concentrations:

no

Details on mating procedure:

Impregnation procedure: Purchased timed pregnant

Duration of treatment / exposure:

From Day 6 up to Day 15 post coitum

Frequency of treatment:

Once daily

Duration of test:

20 d

Dose / conc.:

0 mg/kg bw/day (nominal)

Dose / conc.:

100 mg/kg bw/day (nominal)

Dose / conc.:

300 mg/kg bw/day (nominal)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

30

Control animals:

yes, concurrent vehicle

Details on study design:

Dose selection rationale: Based on the results of toxicological examinations done before (Report No. 486 = TBD 830034, June 27, 1983) (details not reported)

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily

BODY WEIGHT: Yes
- Time schedule for examination: On Day 0, 6, 16 and 20 post coitum

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organs examined: All maternal organs, with emphasis on the uterus and uterine contents

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: Position of fetus in the uterus

Fetal examinations:

External examinations: Yes: [all per litter]
- Soft tissue examinations: Yes: [approximately half per litter]
- Skeletal examinations: Yes: [approximately half per litter]
- Head examinations: Yes: [approximately half per litter]

(See Table 1 for exact number of fetuses examined)

Statistics:

The following statistical methods were used:
If the variables could be assumed to follow a normal distribution, the Dunnett-Test, based on a pooled variance, was applied for the comparison between the treated groups and the control group.
The Steel-Test was applied when the data could not be assumed to follow a normal distribution.
Fisher's Exact test for 2x2 tables was applied if the variables could be dichotomized without loss of information (Bonferroni-Holm-corrected).

Indices:

- Pre-implantation loss (%) = [(Number of corpora lutea - number of implantations)/number of corpora lutea] X 100
- Post-implantation loss (%) = [(Number of implantations - number of live fetuses)/number of implantations] X 100
- Sex ratio (%) = [(number of males/females)/number of fetuses] X 100

Historical control data:

None

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Salivation and propulsion of the head in all dose groups. Additionally, the highest dose group showed a severe salivation. These symptoms were noted variable in the individual groups during the application period.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

No mortality at any dose level.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No treatment-related effects on body weight gain were observed in the dams.

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:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

Placenta and uterus weight: No significant differences between the control and the treatment groups.

Gross pathological findings:

no effects observed

Description (incidence and severity):

No macroscopic changes were observed in the survived dams except for one dam at 100 mg/kg/d bw, which showed greenish-brownish fluid in the uterine horn.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Details on results:

None.

Number of abortions:

not examined

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

Pre-implantation loss was not affected by the treatment. The post-implantation loss and total embryonic deaths were significantly increased in all treatment groups. However, these findings were considered to be incidental (non-treatment-related) because the values in the 100 mg/kg/d bw group were significantly greater than other two higher dose groups and in each group there was one single female with a high incidence of embryonic death.

Total litter losses by resorption:

no effects observed

Early or late resorptions:

not examined

Dead fetuses:

effects observed, non-treatment-related

Description (incidence and severity):

Apart from the control (1 dead foetus) and the 100 mg/kg bw/day groups (7 dead foetuses), all females had viable foetuses. The data for post-implantation loss, embryonic deaths and total foetuses showed some deviations, which were considered to be non-treatment-related.

Changes in pregnancy duration:

not examined

Changes in number of pregnant:

not examined

Other effects:

not examined

Key result

Dose descriptor:

NOAEL

Effect level:

ca. 1 000 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

body weight and weight gain

clinical signs

gross pathology

mortality

organ weights and organ / body weight ratios

Key result

Abnormalities:

no effects observed

Fetal body weight changes:

no effects observed

Description (incidence and severity):

The weights of live fetuses exhibited no significant differences on a litter and individual basis.

Reduction in number of live offspring:

effects observed, non-treatment-related

Description (incidence and severity):

The data for post-implantation loss, embryonic deaths and total foetuses showed some deviations, which were considered to be non-treatment-related.

Changes in sex ratio:

no effects observed

Description (incidence and severity):

The sex ratio of the fetuses was not affected by the treatment.

Changes in litter size and weights:

not examined

Changes in postnatal survival:

not examined

External malformations:

no effects observed

Description (incidence and severity):

No macroscopic findings were observed at external examination of fetuses which were considered to be an effect of the treatment. 1 dead fetus in control and 7 dead fetuses (4 out of 7 partly mummified) in 100 mg/kg/d bw group were recorded. One fetus showed a stump tail at 300 mg/kg/d bw and paleness was observed in one fetus at 1000 mg/kg/d bw. These singular findings are normal observations in the animal strain used.

Skeletal malformations:

no effects observed

Description (incidence and severity):

(i) Retardations: No significant finding at 100 mg/kg/d bw. Two sternebrae were non-ossified in 19 and 29 fetuses (statistically significant) at 300 and 1000 mg/kg/d bw, respectively. Statistically significant increase in the number of fetuses with incomplete ossification of skull bones (17 fetuses) and decrease in the number of fetuses with incomplete ossification of 13th rib (0 fetus) was observed at 1000 mg/kg/d bw. The increased "incomplete ossified skull bones" was essentially due to only 2 dams. The other statistically significant differences were considered to be incidental because these retardation effects were not accompanied by weight retardation and were within the normal range of variation for this strain.
(ii) Variations: No variations in any group.
(iii) Malformations: One fetus with stump tail and missing vertebrae coccigycae at 300 mg/kg/d bw (not considered to be treatment-related).

Visceral malformations:

no effects observed

Description (incidence and severity):

No treatment-related abnormalities.

Other effects:

not examined

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reduction in number of live offspring

changes in sex ratio

fetal/pup body weight changes

external malformations

skeletal malformations

visceral malformations

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

no

Table 2. Summary of performance of mated females

Treatment dose (mg/kg/d)	0	100	300	1000
No. of mated females	30	30	30	30
No. of pregnant females	30	29	28	29
No. of females with premature litter	1	0	2	3
No. of mortalities	0	0	0	0
No. of females with live fetuses at termination	29	29*	26	26

* One dam out of these was not included because the weights of fetuses were not determined

Conclusions:

Under the study conditions, the NOAELs for parental toxicity and developmental toxicity were considered to be 1000 mg/kg bw/day.

Executive summary:

A study was conducted to evaluate the prenatal developmental toxicity of the test substance, C12-18 and C18-unsatd. DEA, according to OECD Guideline 414, in compliance with GLP. The substance was administered to groups of 30 female rats by gavage at dose levels of 0, 100, 300 and 1000 mg/kg bw/day, once daily from Gestation Days (GD) 6 to 15 inclusive. Control animals were dosed with the vehicle alone (arachis oil, DAB 9). Clinical condition and reaction to treatment were recorded at least once daily. Body weights were reported on GD 0, 6, 16 and 20. All surviving females were sacrificed on GD 20 and the foetuses were removed by caesarean section. At necropsy, the females were examined macroscopically. Live foetuses were weighed, sexed and examined for visceral and skeletal abnormalities. No deaths or treatment-related changes in body weight gain and necropsy findings were observed in dams at any dose level. Treatment-related symptoms observed in all groups were salivation and propulsion of the head. The highest dose group showed severe salivation. Apart from the control (1 dead foetus) and the 100 mg/kg bw/day groups (7 dead foetuses), all females had viable foetuses. Pre-implantation loss and mean numbers of resorptions were not affected by treatment. The data for post-implantation loss, embryonic deaths and total foetuses showed some deviations, which were considered to be non-treatment-related. Mean placental and uterus weights were not affected by the treatment. Foetal sex ratio was comparable in all groups. No treatment-related foetal abnormalities were found at necropsy. The examined foetuses showed no treatment-related visceral and skeletal abnormalities/variations. One foetus at 300 mg/kg bw/day showed a stump tail and missing coccigycae vertebrae. Further, the data for skeletal ossifications showed some deviations in the two highest dose groups. However, all these effects were assessed to be non-treatment-related. Under the study conditions, the NOAELs for parental toxicity and developmental toxicity were considered to be 1000 mg/kg bw/day (Pitterman, 1994).

Reference 2

Endpoint:

developmental toxicity

Type of information:

read-across based on grouping of substances (category approach)

Adequacy of study:

key study

Study period:

As of 2021 at the earliest. Depending on the results of the other planned studies (e.g. OECD TG 421, 408) and testing proposal acceptance.

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

KL2 due to RA

Justification for type of information:

TESTING PROPOSAL ON VERTEBRATE ANIMALS

NON-CONFIDENTIAL NAME OF SUBSTANCE:
- Amides, C8-18 (even numbered) and C18-unsatd., N,N-bis(hydroxyethyl (C8-18 and C18-unsatd. DEA), EC No. 931-329-6

CONSIDERATIONS THAT THE GENERAL ADAPTATION POSSIBILITIES OF ANNEX XI OF THE REACH REGULATION ARE NOT ADEQUATE TO GENERATE THE NECESSARY INFORMATION [please address all points below]:
- Available GLP studies
: none
- Available non-GLP studies
: none
- Historical human data
: none
- (Q)SAR
: not applicable
- In vitro methods
: not applicable
- Weight of evidence
: not sufficient
- Grouping and read-across
: not sufficient
- Substance-tailored exposure driven testing [if applicable]
: none
- Approaches in addition to above [if applicable] : not applicable
- Other reasons [if applicable]
: none

CONSIDERATIONS THAT THE SPECIFIC ADAPTATION POSSIBILITIES OF ANNEXES VI TO X (AND COLUMN 2 THEREOF) OF THE REACH REGULATION ARE NOT ADEQUATE TO GENERATE THE NECESSARY INFORMATION:
- Proposed adaptations were not considered sufficient to address this endpoint. This was discussed with ECHA in the frame of a Dossier Improvement Action Plan (DIAP).

FURTHER INFORMATION ON TESTING PROPOSAL IN ADDITION TO INFORMATION PROVIDED IN THE MATERIALS AND METHODS SECTION:
- Proposed study design: according to OECD Guideline 414

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

GLP compliance:

yes (incl. QA statement)

Species:

rabbit

Route of administration:

oral: gavage

Dose descriptor:

NOAEL

Remarks on result:

other: Testing planned

Abnormalities:

not specified

Dose descriptor:

NOAEL

Remarks on result:

other: Testing planned

Abnormalities:

not specified

Developmental effects observed:

not specified

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

Sufficient data available.

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

A study was conducted to evaluate the prenatal developmental toxicity of the C12-18 and C18-unsatd. DEA, according to OECD Guideline 414, in compliance with GLP. The substance was administered to groups of 30 female rats by gavage at dose levels of 0, 100, 300 and 1000 mg/kg bw/day, once daily from Gestation Days (GD) 6 to 15 inclusive. Control animals were dosed with the vehicle alone (arachis oil, DAB 9). Clinical condition and reaction to treatment were recorded at least once daily. Body weights were reported on GD 0, 6, 16 and 20. All surviving females were sacrificed on GD 20 and the foetuses were removed by caesarean section. At necropsy, the females were examined macroscopically. Live foetuses were weighed, sexed and examined for visceral and skeletal abnormalities. No deaths or treatment-related changes in body weight gain and necropsy findings were observed in dams at any dose level. Treatment-related symptoms observed in all groups were salivation and propulsion of the head. The highest dose group showed severe salivation. Apart from the control (1 dead foetus) and the 100 mg/kg bw/day groups (7 dead foetuses), all females had viable foetuses. Pre-implantation loss and mean numbers of resorptions were not affected by treatment. The data for post-implantation loss, embryonic deaths and total foetuses showed some deviations, which were considered to be non-treatment-related. Mean placental and uterus weights were not affected by the treatment. Foetal sex ratio was comparable in all groups. No treatment-related foetal abnormalities were found at necropsy. The examined foetuses showed no treatment-related visceral and skeletal abnormalities/variations. One foetus at 300 mg/kg bw/day showed a stump tail and missing coccigycae vertebrae. Further, the data for skeletal ossifications showed some deviations in the two highest dose groups. However, all these effects were assessed to be non-treatment-related. Under the study conditions, the NOAELs for parental toxicity and developmental toxicity were considered to be 1000 mg/kg bw/day (Pitterman, 1994).  

In addition, after discussion with ECHA in the frame of a Dossier Improvement Action Plan (DIAP), a testing proposal is submitted for the conduct of a prenatal developmental toxicity study in rabbit according to OECD Guideline 414 with the read across substance, C8-18 and C18-unsatd. DEA.

Additional considerations

Based on an in-depth analysis of the available information (see read-across justificationin Section 13 of the IUCLID dataset), it is the FAA consortium’s hypothesis that a read-across within and across MEA, DEA and MIPA derived alkanolamides is scientifically plausible and justified. While there is at this point no evidence putting the read-across hypothesis in question, the FAA consortium recognizes some limitations, predominantly related to the quality of individual endpoint studies (including quality of the test substance characterisations) and existing higher Tier endpoint data gaps. After discussion with ECHA in the frame of a Dossier Improvement Action Plan (DIAP), the FAA Consortium agrees to the need to strengthen the toxicology data-based link between and within the DEA, MEA and MIPA subcategories.

In view of this, the FAA Consortium decided to proceed with a 3-Tier testing strategy. In Tier 1, a series of bridging studies according to OECD TG 422 will be conducted with a representative short- and a long chain substance of each subcategory (i.e., DEA, MEA, MIPA).

The objective of Tier 2 will be to generate a complete set of higher toxicology data for a selected >1000 tpa substance (i.e., OECD TG 408/443/414 (rats)/414 (2cnd species). The testing in Tier 2 will be conducted with C8-18 and C18-unsatd. DEA, the substance that is generally perceived to be the most critical from a reproductive toxicity point of view based on the classification of DEA.

Tier 1 and 2 proposed studies have been included in the present dossier update. Should these suggestsignificant differences in type and strength of effects between theDEA, MEA and MIPA subcategories, therefore not supporting the current read across justification, further testing may be initiated. The Consortium’s strategy is detailed in a document entitled ‘ECHA-DIAP - FAA testing strategy summary – 24Sep20’ attached in Section 13 of the IUCLID dataset.

Justification for classification or non-classification

The available data suggests that the test substance is not a reproductive toxicant with regard to fertility or developmental effects. However, the registered compositions indicate that some of the test substances may contain ≥3% free DEA. This constituent has now been self-classified by its REACH registrants as Repr. 2 -H361: Suspected of damaging fertility or the unborn child(https://echa.europa.eu/registration-dossier/-/registered-dossier/15770/2/1/?documentUUID=b015c4bf-61f0-4b7b-b858-cfe6f686f088) according to CLP (EC 1272/2008) criteria.Therefore, based on the mixture classification rules, the test substance warrants no classification for reproductive or developmental toxicity when its free DEA content is <3% but classification as Repr. 2 -H361: Suspected of damaging fertility or the unborn child when the free DEA content is ≥3%.