Carbon black

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

A relevant systemic exposure after oral, dermal or respiratory administration is not expected (see section on toxicokinetics for details). Because a lower bioavailability is expected for the non-nanoforms as compared to the nanoforms and since carbon black, irrespective of form display a similar surface chemistry (the functional groups on surface of their particles are essentially the same but may differ in concentration), differences in toxicological outcome is not expected. Hence the data from the not treated nanoforms are read-across to the non-nanoform (for details on key information, see summaries for nanoforms) 

Link to relevant study records

Reference

Reference 1

Endpoint:

multi-generation reproductive toxicity

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Justification for type of information:

Endpoint-specific read-across justification" attached under section 13.2

Reason / purpose for cross-reference:

read-across source

Qualifier:

no guideline followed

Principles of method if other than guideline:

Time-mated females were exposed to a mean total suspended particle mass concentration of 4.79 ± 1.86 or 33.87 ± 14.77 mg/m3 for the low and high exposure, respectively. In this study, male fertility parameters were assessed following in utero inhalation exposure to carbon black in four generations of mice.

GLP compliance:

not specified

Remarks:

Study is published in open literature

Limit test:

no

Species:

mouse

Strain:

NMRI

Sex:

male

Analytical verification of doses or concentrations:

yes

Description (incidence and severity):

no effects on testosterone levels

Reproductive function: sperm measures:

no effects observed

Dose descriptor:

NOEC

Effect level:

34 mg/m³ air

Based on:

test mat.

Sex:

female

Basis for effect level:

clinical signs

mortality

Key result

Dose descriptor:

NOAEC

Effect level:

> 34 mg/m³ air

Based on:

test mat.

Sex:

female

Basis for effect level:

clinical signs

mortality

Key result

Critical effects observed:

no

Dose descriptor:

NOEC

Effect level:

34 mg/m³ air

Based on:

test mat.

Sex:

male

Basis for effect level:

reproductive function (sperm measures)

Key result

Dose descriptor:

NOAEC

Effect level:

> 34 mg/m³ air

Based on:

test mat.

Sex:

male

Basis for effect level:

reproductive function (sperm measures)

Key result

Critical effects observed:

no

Dose descriptor:

NOEC

Generation:

F1

Effect level:

34 mg/m³ air

Based on:

test mat.

Sex:

male

Basis for effect level:

other: reproductive function

Key result

Dose descriptor:

NOAEC

Generation:

F1

Effect level:

> 34 mg/m³ air

Based on:

test mat.

Sex:

male

Basis for effect level:

other: reproductive function

Key result

Critical effects observed:

no

Gross pathological findings:

no effects observed

Key result

Dose descriptor:

NOAEC

Generation:

F2

Effect level:

> 34 mg/L air (analytical)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: sperm parameters

Key result

Critical effects observed:

no

Key result

Reproductive effects observed:

no

Conclusions:

In utero exposure to carbon black via maternal whole body inhalation did not affect male-specific reproductive, fertility and litter parameters in four generations of mice.

Executive summary:

Time-mated females were exposed to a mean total suspended particle mass concentration of 4.79 ± 1.86 or 33.87 ± 14.77 mg/m3 for the low and high exposure, respectively. Exposure did not affect gestation and litter parameters in any generation. No significant changes were observed in body and reproductive organ weights, epididymal sperm parameters, daily sperm production, plasma testosterone or fertility.

Reference 2

Endpoint:

multi-generation reproductive toxicity

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Justification for type of information:

Endpoint-specific read-across justification" attached under section 13.2

Reason / purpose for cross-reference:

read-across source

Key result

Dose descriptor:

NOEL

Effect level:

268 other: µg/animal/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

Remarks on result:

other: dosed on gd 7, 10, 15 and 18

Key result

Critical effects observed:

no

Key result

Dose descriptor:

NOEC

Effect level:

268 other: µg/animal

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: number of offfspring, sex ratio

Key result

Dose descriptor:

NOEC

Generation:

F1

Effect level:

268 other: µg/animal

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: number of offspring and sex ratio

Key result

Critical effects observed:

no

Key result

Dose descriptor:

NOEC

Generation:

F2

Effect level:

268 other: µg/animal

Based on:

test mat.

Sex:

female

Basis for effect level:

other: no effects on sex ratio, number of offspring, ESTR mutations

Key result

Critical effects observed:

no

Key result

Reproductive effects observed:

no

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Effect on fertility: via inhalation route

Endpoint conclusion:

no adverse effect observed

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Effects on developmental toxicity

Description of key information

A relevant systemic exposure after oral, dermal or respiratory administration is not expected (see section on toxicokinetics for details). Because a lower bioavailability, if any, is expected for the non-nanoforms as compared to the nanoforms and since carbon black, irrespective of form display a similar surface chemistry; the functional groups on surface of their particles are essentially the same but may differ in concentration, differences in toxicological outcome is not expected. Hence the data from the nano forms are read-across to the non nano form (for details on key information, see summaries for nanoforms) 

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

Type of information:

experimental study planned (based on read-across)

Justification for type of information:

TESTING PROPOSAL ON VERTEBRATE ANIMALS
Prenatal developmental toxicity study according to OECD TG 414 in rabbits

NON-CONFIDENTIAL NAME OF SUBSTANCE:
-Name of the substance on which testing is proposed to be carried out: Carbon black, Cas No. 1333-86-4

CONSIDERATIONS THAT THE GENERAL ADAPTATION POSSIBILITIES OF ANNEX XI OF THE REACH REGULATION ARE NOT ADEQUATE TO GENERATE THE NECESSARY INFORMATION

- Available GLP studies
Tests in Rabbit: No GLP study available.

Tests in rats:
A prenatal developmental toxicity study in rats via gavage was conducted under GLP using a standard OECD protocol (OECD 414;). Pregnant female rats were dosed through daily oral gavage at 0, 100, 300 or 1000 mg/kg/day (24/group) during the sensitive period of organogenesis [daily through days 5 to 19 of gestation]. The oral administration of carbon black to pregnant rats during the sensitive period of organogenesis was well-tolerated up to the highest dose tested, and there were no adverse maternal changes or any effects on embryo-foetal development. Accordingly, under the conditions of this study, the No Observed Adverse Effect Level (NOAEL) was 1000 mg/kg bw/day.
Further, a repeated dose oral toxicity study in accordance with OECD guideline 408 is available for carbon black. Repeated daily oral (gavage) administration of carbon black to rats for 13 weeks at 100, 300 or 1000 mg/kg/day was well-tolerated, with some non-adverse findings related to the staining properties of carbon black. Importantly, no adverse effects on reproductive organs or tissues or other concerns in relation to reproductive toxicity were identified. Accordingly, under the conditions of this study, the No Observed Adverse Effect Level (NOAEL) was 1000 mg/kg bw/day.

- Available non-GLP studies
Jackson et al. (2012) assessed the effects on the development of the offspring in mice following maternal exposure of time-mated C57BL/6BomTac mice to ca. 42 mg/m3 Printex 90 for 1 h/day on gestation days (GD) 8–18. Effects on gestation, lactation and DNA strand breaks were evaluated. Traditional gestational and litter parameters were measured. Inflammation was assessed in maternal bronchoalveolar lavage (BAL) after exposure, and at weaning. Levels of DNA strand breaks were assessed in maternal BAL cells and liver, and in offspring liver. Dams were monitored until weaning and some offspring until adolescence. Visceral and skeletal effects in offspring were not studied. Persistent inflammation in the lung was recorded in dams. Gestational and litter parameters in treated dams and their offspring were similar to that of control animals. Results for weight gain during gestation and lactation, gestation length, offspring weight at birth, during lactation and maturation, litter size, gender ratio, number of implantations, and post-natal viability were unremarkable when compared to controls. A NOAEC for maternal toxicity cannot be derived as marked Inflammation in the lungs of dams were noted which lasted until the end of lactation. Based on assessed parameters in offspring, a NOEC for developmental toxicity of 42 mg/m3 can be assigned. In a four-generation study investigating the effects of carbon black on the reproductive function of male mice, inhalation exposure of time-mated female mice to 4.79 ± 1.86 mg/m3 or 33.87 ± 14.77 mg/m3 did not affect gestational (gestation length, number and loss of implantations) and litter parameters (offspring weight, litter size, sex ratio) in any generation. No significant changes were observed in body and reproductive organ weights, epididymal sperm parameters, daily sperm production, plasma testosterone or fertility. Visceral and skeletal effects in offspring were not studied. Under the conditions of this study; a NOEC for developmental toxicity of ca. 34 mg/m3 can be assigned (Skovmand et al. 2019). This study does not provide information equal to that of a PNDTstudy and as such does not qualify for use to meet the information requirements for a second species.

- Historical human data: not available
- (Q)SAR: not available
- In vitro methods: not available
- Weight of evidence

At the Annex X level (registration at 1000 or more tonnes per year), provision of PNDT information in two species is the standard information requirement. An oral PNDT study conducted with carbon black under GLP and in accordance with OECD 414 reported no adverse maternal changes and no effects on embryo-foetal development at the highest dose tested (NOAEL 1000 mg/kg bw/day). Furthermore, in a non-GLP study in mice, no gestational or developmental effects were reported in a whole-body inhalation study at 42 mg/m3. Information from the available GLP studies do not indicate any triggers for developmental toxicity.

Information in a second species, preferably the rabbit, is lacking and therefore needs to be generated to comply with the data requirements under REACH for the relevant tonnage band for carbon black.

- Grouping and read-across:
Carbon black is a unique chemical substance that cannot be considered to be part of any group of chemically similar substances from which read across can be applied.


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:

Amongst others, Column II of Annex X allows that a PNDT study in a second species must not be conducted if a substance is of low toxicological activity, no (or low) systemic availability and there is no or no significant human exposure.
Carbon black is a substance of low toxicological activity, and available toxicokinetic (TK) data indicates that systemic absorption through skin, via ingestion or pulmonary exposure is insignificant. This conclusion is corroborated by the consistent lack of adverse systemic effects in available oral and inhalation studies for carbon black. The lack of systemic availability of carbon black has been demonstrated in TK studies with adult male and female rats using two types of carbon black (Be7-labelled) representing the two sets of nanoforms covered by the dossier; carbon black “not treated”, and carbon black “treated” (with oxidizing agents). In brief, the modified OECD 417 TK study involved both intratracheal instillation and oral (gavage) application of carbon black in rats, and was designed to understand whether carbon black is absorbed into the body and can reach various organs, in particular, reproductive organs in female and male rats (an additional module of male rats was included for the specific purpose of testing whether radiolabelled carbon black is observed in reproductive organs in male rats). For both the instillation and oral routes of exposure, no systemic absorption of carbon black was observed. The results of these investigations confirm the poor absorption and bioavailability of carbon black (Fraunhofer 2021). The findings of this study in rats agree with those of an earlier oral toxicokinetic study by LeFevre et al (1986) in mice. Using 7Be-labeled carbon black (Elftex 8, Cabot Corporation), the authors showed that (less than 0.014% of the applied radioactivity was found in non-intestinal tissues of mice after a single oral exposure. The distribution of the isotope was determined in the animals 4 hours and 1, 2, 5 and 14 days after exposure. In Peyer’s patches, less than 0.002% and 0.006% radioactivity were found in young and aged mice, respectively. According to the authors, the radioactivity in extra intestinal viscera and blood was negligibly low and practically all the label was excreted in the feces (LeFevre and Joel 1986).
Even though carbon black meets the criteria of low toxicological activity and no (or low) systemic availability, exposure to some worker populations, although well controlled and below OELs set for CB, cannot be considered as insignificant. Therefore, conditions for waiving of a PNDT study in second species as laid down in column 2 of Annex X are not met fully.

Route of administration
REACH stipulates that reproductive toxicity studies shall be conducted via the most appropriate route of administration, having regard to the likely route of human exposure. Within the context of REACH, the likely routes of human exposure are: oral, inhalation and dermal routes.

Further to this, ECHA guidance (ECHA R7a) states that the most appropriate and default route of administration depends on the most appropriate route for identification of the intrinsic properties of the substance under investigation. The default and preferred route for the identification of reproduction toxicity is the oral route because it can reasonably be seen as allowing for a maximization of the systemic availability (internal dose) for most substances. ECHA guidance on reproduction toxicity testing also stipulates that especially for dusts or particulate substances, the default and preferred route of administration in reproductive toxicity testing is the oral route; a deviation from which must be justified by the registrant (ECHA R7a). The Lead Registrant (LR) agrees with this conclusion and contends that the internal dose of carbon black is more likely to be maximized via oral exposure (e.g., by testing up to the limit dose) as opposed to the inhalation route which is limited intrinsically due to the onset of lung overload effects occurring already at comparatively low inhalation exposure levels and characterised by severe inflammatory reactions and impairment of normal physiological function of respiratory organs. Furthermore, available toxicological information for carbon black does not indicate that route-specific effects on reproduction or differences in toxicokinetics are to be expected which would support a deviation from the default oral route. Therefore, the LR contends that for carbon black and for the purpose of this study, the oral route is most appropriate to identify potential systemic effects, as the oral route will increase the potentially greater bioavailability of the test substance.

This conclusion is further substantiated by the following scientific reasoning:

MPPD modelling of fractional deposition in the respiratory tract upon inhalation clearly demonstrates that when rats and humans are exposed to airborne particles of carbon black, the overwhelming majority of material deposited in the respiratory tract would be translocated to the gastrointestinal tract and not to the pulmonary region; the ratio of inhalable material translocated to the human GI tract compared to the pulmonary region of the human lung being approx. 60:3 (model calculations provided by EBRC Consulting GmbH 2019).The underlying data can be summarised as follows:

A candidate carbon black test item (Printex 90) was subjected to dustiness testing according to the modified Heubach rotating drum method which includes the determination of the airborne particle size distribution by an adjacent 8-stage cascade impactor measurement (Schamberg & Gogilan 2019). The airborne material showed a bimodal particle size distribution (P1 0.26; MMAD1 0.8 and GSD1 1.66: P2 0.74; MMAD2 13.56 and GSD2 2.36). The results on dustiness testing are corroborated by actual workplace PSD data (Tsai et al. 2001) for workers at a carbon black manufacturing facility. The exposure of workers in the pelletising, as well as the final packaging area of a carbon black manufacturing facility, was characterised both for personal exposures (using the standard IOM sampler to monitor the inhalable fraction) and particle size distribution (by 9-stage MOUDI and 4-stage NRI samplers). The MOUDI particle size distribution yielded a bimodal distribution with a smaller peak at MMAD = 0.3 μm and a larger peak representing the majority of airborne material at MMAD = 21-23 µm. Kuhlbusch et al. (2004 & 2006) also reported a bimodal particle size distribution of airborne carbon black with the particles of aerodynamic diameters > 0.4 µm. These results indicate that, upon mechanical agitation, only a sub-fraction of the particles has the propensity to become airborne and inhaled.
Based on the airborne particle size distribution of carbon black (Printex 90), the total deposition in the entire human respiratory tract can be predicted with the MPPD model to be approx. 64% of the inhalable material. A sub-categorisation for the different regions of the respiratory tract, revealed that only a small sub-fraction (approx. 3%) of the inhalable particles will deposit in the pulmonary region of the respiratory tract, whereas the remaining portion is predicted to deposit in the tracheobronchial (TB; approx. 1.73%) and extra-thoracic regions (head; approx. 59.1%). Thus, the overwhelming majority of inhaled particles will be deposited in the upper regions of the respiratory tract, of which almost 95% will subsequently be cleared rapidly by translocation to the gastrointestinal tract.
This MPPD model prediction is validated by empirical rat data from Mauderly et al. 1994; rats were subjected to counts of radioactivity to determine the amounts of 7Be present at 0, 4, 7, 13, 28, 35, 42, 56, 73, 84, 98, 112, and 126 days after nose only inhalation exposure to [7Be]-labelled carbon black in male and female rats. They reported that by day 7, radioactivity in the head, large airways, and gastrointestinal tract (GIT) was already negligible due to early mucociliary clearance from the lung and faecal excretion. Southam et al (2002) quantified the relative distribution achieved with intranasal delivery of a non-absorbable tracer, 99mTc-labeled sulphide colloid in upper and lower respiratory tract (URT and LRT) in mice. Over the course of 1 h following instillation of 50 µl of the tracer colloids, radiation in the URT decreased and appeared in the GIT (Southam et al. 2002).

Whereas ECHA guidance R7a states that testing by the inhalation route is appropriate when testing nanomaterials for certain endpoints like repeated dose toxicity if human inhalation exposure is likely, the Lead Registrant submits that the possibility of exposure to particles of an inhalable size (aerodynamic diameter below 100 μm) has to be viewed in combination with their deposition kinetics in the human lung. The modelling and animal data underscore this point vividly considering the much lower amounts of inhaled particles that are predicted to reach the alveolar regions in comparison to that which is present in the gut upon direct oral application (up to 1000 mg/kg bw). It thus becomes evident that the potential to maximize the internal dose is far higher via the oral than the inhalation route.

In addition, studies via the inhalation route and their interpretation are inherently limited due to alveolar macrophage overload effects and the corresponding impairment of respiratory function that occur at high concentrations of particles. In an inhalation study with a poorly soluble substance like carbon black, it can be expected that lung overload-inducing concentrations will be attained already at comparatively low exposure levels, thereby precluding the potential to fulfil the guideline requirement to induce some level of systemic developmental and/or maternal toxicity (clinical signs or a decrease in body weight) at the mid and highest tested concentrations.

An Expert workshop on lung overload recently concluded that in principle, lung overload can occur in all species (Driscoll and Borm 2020). Given that lung overload constitutes a stressful situation for the animal, it is therefore likely that lung overload induced stress in the pregnant animals may influence study outcome and hamper results interpretation. In any case, the Lead Registrant notes that the rabbit is particularly sensitive and susceptible to stress situations, and that stress during pregnancy can result in prenatal loss and negatively influence test results. The OECD TG 414 recommends therefore that stress of dams be avoided at all costs to guard against effects that are not treatment-related. As indicated in the previous paragraph, poorly soluble particles like carbon black (a) will cause lung overload already in rodents at comparatively low exposure levels, (b) must be administered at high concentrations in order to elicit a measurable response in maternal toxicity and (c) when administered at high concentrations will cause lung overload and a dyspnoeic response which are additional unnecessary stressors for the animals.

Further to the above, following initial discussions with a number of contract testing laboratories, the Lead Registrant has learned that inhalation studies in rabbits have not been, and are still not being, routinely performed with particulate substances. It is noted that ECHA’s Board of Appeal ruled in case number A-0005-2011 on the unprecedented nature of repeated dose testing via the inhalation route in rabbits. Although inhalation PNDT studies in rabbit are available for gaseous substances, the Lead Registrant could identify no published study in the past 20 years for any particulate substance. The lack of essential historical control data for the inhalation route would impede the evaluation of effects in terms of toxicological relevance (substance-related versus incidental occurring). The Lead Registrant therefore contends that the logic underlying the decisions of case number A-0005-2011 apply to PNDT testing via the inhalation route and that testing via the oral route will yield more meaningful results for the reasons set forth further above in detail.

Considering all the information presented above, the Lead Registrant therefore proposes to conduct the proposed PNDT study on the effects of carbon black in the rabbit via the oral route.

Test Material:
The Lead Registrant proposes to test Printex 90, a nanoform of the set for carbon black (solid: nanoform, no surface treatment). Based on the arguments provided in the justification for grouping of similar nanoforms, the Lead Registrant submits that Printex 90 is the most suitable representative for testing. This will facilitate a meaningful comparison with existing animal toxicity data, since many of the toxicity studies published in the open literature have been performed with this grade of carbon black. Based on the arguments provided in the dossier to justify read across of data between sets of similar nanoforms, the Lead Registrant submits the study performed with this untreated nanoform is suitable for the identification of hazardous potential of other forms of carbon black in the registration dossier.

Duration of study
There is no existing data on the tolerance of carbon black in the rabbit. Therefore, there is a need to first ascertain the maximum tolerated dose in non-pregnant rabbits. Exploratory discussions with experienced contract laboratories indicate that an estimated timeline of 12 - 15 months is required.

References
Kevin E. Driscoll & Paul J. A. Borm (2020): Expert workshop on the hazards and risks of poorly soluble low toxicity particles. Inhalation Toxicology. https://doi.org/10.1080/08958378.2020.1735581
Kuhlbusch TAJ, Neumann S., Fissan H: Number Size Distribution, Mass Concentration, and Particle Composition of PM1, PM2.5, and PM10 in Bag Filling Areas of Carbon Black Production. Journal of Occupational and Environmental Hygiene, 1: 660–671
Kuhlbusch TAJ, and Fissan H: Particle Characteristics in the Reactor and Pelletizing Areas of Carbon Black Production. Journal of Occupational and Environmental Hygiene, 3: 558–567
Southam DS, Dolovich M, O’Byrne PM and Inman MD. Distribution of intranasal instillations in mice:effects of volume, time, body position, and anesthesia. Am J Physiol Lung Cell Mol Physiol 282: L833–L839, 2002
Tsai, Perng-Jy, Hong-Yong Shieh, Wen-Jhy Lee, and Soon-Onn Lai. 2001. "Characteristics of Exposure Profiles for Workers Exposed to Airborne Dusts and Polycyclic Aromatic Hydrocarbons (PAHs) in the Carbon Black Manufacturing Industry." Journal of Occupational Health 43 (3):118-128. doi: 10.1539/joh.43.118.
Skovmand, Astrid, Alexander C. Ø Jensen, Clotilde Maurice, Francesco Marchetti, Anna J. Lauvås, Ismo K. Koponen, Keld A. Jensen, Sandra Goericke-Pesch, Ulla Vogel, and Karin S. Hougaard. 2019. "Effects of maternal inhalation of carbon black nanoparticles on reproductive and fertility parameters in a four-generation study of male mice." Particle and fibre toxicology 16 (1): 13-13. https://doi.org/10.1186/s12989-019-0295-3. https://pubmed.ncbi.nlm.nih.gov/30879468. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6421671/.


FURTHER INFORMATION ON TESTING PROPOSAL IN ADDITION TO INFORMATION PROVIDED IN THE MATERIALS AND METHODS SECTION.

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rabbit

Strain:

New Zealand White

Route of administration:

oral: gavage

Vehicle:

other: HPMC or similar

Analytical verification of doses or concentrations:

yes

Control animals:

yes, concurrent vehicle

Reference 2

Endpoint:

developmental toxicity

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Justification for type of information:

Endpoint-specific read-across justification" attached under section 13.2

Reason / purpose for cross-reference:

read-across source

Species:

mouse

Route of administration:

inhalation

Key result

Dose descriptor:

LOEC

Effect level:

41.7 mg/m³ air

Based on:

test mat.

Basis for effect level:

other: inflammatory markers in BAL increased; persisted until the end of lactation (weaning) as evidenced in the increase in the number of neutrophils and lymphocytes and DNA damage registered in the livers of dams

Key result

Abnormalities:

no effects observed

Key result

Dose descriptor:

NOEC

Effect level:

41.7 mg/m³ air

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

changes in postnatal survival

other: gestational and deveopmental toxicity

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

no

Executive summary:

In an inhalation study, carbon black was administered to 22 time-mated pregnant C57BL/ 6/BomTac mice at a single concentration of ca. 41.7 mg/m3 from days 8 through 18 of gestation. Twenty-two control animals were exposed concomitantly to air. Traditional gestational and post-gestational parameters were assessed in dams and their offspring. In addition, the investigators sought to answer the question whether inhalation exposure to carbon black caused DNA damage in lung (BAL) and liver cells of exposed dams as well as in the liver of their offspring. Lung inflammation in dams was also monitored. Samples for analysis of DNA damage and lung inflammation were collected 5 and 24 days after cessation of exposure in dams and on post-natal days 2, 22-23 and 50 for DNA analysis in offspring.

Gestational and litter parameters in exposed dams and their offspring were similar to controls. Results for weight gain during gestation and lactation, gestation length, offspring weight at birth, during lactation and maturation, litter size, gender ratio, number of implantations, and post-natal viability were unspectacular when compared to controls.

No effects on DNA strand breaks were noted in BAL fluid cell at any investigation time point. Increased level of DNA damage was observed in the livers of exposed dams at both sampling time points of 5 and 24 days after exposure. In the offspring, the level of DNA strand breaks was higher in liver at weaning and in adolescents, compared to their controls (weaning: 1.4-fold increase, p = 0.001; adolescents: 1.5-fold increase p = 0.011). The level of oxidatively generated DNA damage in the liver of offspring was also determined by the level of formamidopyrimidine DNA glycosylase (FPG) enzyme sensitive sites. There was no consistent increase in oxidatively generated DNA damage in the offspring liver cells at any sampling time point i.e. PND 2 (newborn), PND 22-23 (weaning) or PND 50 (adolescent) (newborn; exposed 0.91 ± 0.27 vs. control 0.71 ± 0.20; weaning exposed 1.05 ± 0.12 vs. control 1.28 ± 0.13; adolescents exposed 0.87 ± 0.10 vs. control 1.20 ± 0.11. All data are presented as lesions per 10⁶base pairs).

BAL analysis indicated the presence of persistent inflammation in the lungs of time-mated mice; evidenced in the increased level of neutrophils in BALfluid compared to their controls 5 and 24 days after exposure (5 days: 11.4-fold increase,p=0.008; 24 days: 11.6-fold increase,p<0.001). Also recorded were mildly increased levels of lymphocytes in BALfluid 5 days after exposure (3.4-fold increase,p=0.020) and total cell counts at both time-points (5 days: 1.5-fold increase,p=0.032; 24 days: 1.2-fold increase,p=0.057).

Reference 3

Endpoint:

developmental toxicity

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Justification for type of information:

Endpoint-specific read-across justification" attached under section 13.2

Reason / purpose for cross-reference:

read-across source

Species:

rat

Route of administration:

oral: gavage

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Remarks on result:

other: discoloration of faeces was not considered an adverse effect

Key result

Abnormalities:

no effects observed

Key result

Dose descriptor:

NOEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects reported

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

no

Conclusions:

The oral No Observed Adverse Effect Level (NOAEL) for maternal toxicity and the No Observed Effect Level (NOEL) for developmental toxicity were both set at 1000 mg/kg bw/day in a study evaluated by the SCCS

Executive summary:

The SCCS summarized the study and reports that the study authors concluded that oral administration of carbon black to pregnant rats at 100, 300 or 1 000 mg/kg bw/d during the sensitive period of organogenesis was well tolerated. There were no adverse maternal changes or any effects on embryo-fetal development. Accordingly, under the conditions of this study, the No Observed Adverse Effect Level (NOAEL) for maternal toxicity and the No Observed Effect Level (NOEL) for developmental toxicity were both set at 1000 mg/kg bw/day. This study is used in a weight of evidence assessment for the developmental toxicity assessment. Together with modelled dataon deposition fractions in the lungs of rats and humans it becomes evident that a significant amount of carbon black particles inhaled by rats and humans will be cleared from the lungs into the gastrointestinal tract; making this route of exposure of relevance for the human risk assessment.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no adverse effect observed

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Toxicity to reproduction: other studies

Description of key information

No relevant information available.

Mode of Action Analysis / Human Relevance Framework

see set_not treated

Justification for classification or non-classification

The criteria for classification according to the EU CLP Regulation are not met.